Patent Application
20220226385
The present invention relates to a pharmaceutical composition containing vitrified periodontal tissue-derived mesenchymal stem cells for prevention or treatment of male infertility.
The number of male infertility patients has increased rapidly over the past four years, increasing about 1.5 times from 43,094 in 2013 to 62,468 in 2017. As of 2012, the number of males aged 35 to 39 who had undergone infertility treatment per year was 695 per 100,000 of population and the number of males aged 40 to 44 was 266 per 100,000. In a gender comparison over the past five years, the number of female infertility patients has increased by an average of 2.5% per year, while the number of male infertility patients has rapidly increased by 11.8% per year.
Male infertility is caused by various factors, including hormonal problems including male hormones, testicular problems due to exposure to toxic substances, infection, and trauma, problems with sperm, and abnormal passageways of sperm release. Recently, it has been reported that male infertility shows a higher than average increase rate in relation to modern lifestyles such as obesity, diabetes, smoking and stress. Obesity was overlooked until early 2010, and as a pathological association with infertility has been reported, it has recently attracted attention as a factor directly related to infertility among numerous factors of infertility. A series of studies have been reported that in obese men, excessive adipose tissue causes problems in the metabolism of sex hormones, which changes the male hormone (testosterone) to the female hormone (estrogen) and interferes with sperm production, and excess fat in obese men raises body temperature, and thus sperm cannot be produced well. In addition, non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and the main cause is known to be obesity. NAFLD causes sex hormone imbalance due to hepatocyte damage, leading to chronic male infertility such as male hypogonadism. In particular, since most estrogen is metabolized in the liver tissue, damage to hepatocytes causes a problem in the estrogen removal system, which has been proven by studies that sex hormone imbalance due to liver cirrhosis is improved after liver transplantation.
As for male infertility, which is related to obesity, liver disease, and the like, hormone therapy, and various drugs that can affect spermatogenic function are used, such as kallikrein, carnitine, pentoxifylline, nonsteroidal anti-inflammatory drugs, and alpha blockers. In the case of hormone therapy such as testosterone replacement therapy (TRT), the clinical success rate is inconsistent or fatal side effects are reported. In the case of non-hormone therapy, the exact standard of action of a drug is often unknown. In addition, it is known that there is no effective treatment method for hypogonadism. Accordingly, there is a need for new therapies that can fundamentally treat chronic male infertility due to hepatocyte damage caused by obesity and NAFLD.
Periodontal tissue-derived mesenchymal stem cells are a type of mesenchymal stem cells. According to recent studies, it has been found that mesenchymal stem cells can differentiate not only into mesoderm but also into ectoderm (mainly nerve cells) or endoderm (hepatocytes, pancreatic cells, etc.), and are recognized as having multipotency.
Accordingly, the present inventors have tried to develop a new therapeutic agent that can replace male infertility treatment, which has many side effects reported, and as a result, the present inventors found that when vitrified periodontal tissue-derived mesenchymal stem cells were administered to an animal model of infertility through a high-fat diet, sex hormone levels in seminiferous tubules of testes and blood were recovered to levels similar to those of normal models, thereby being effective against male infertility, and then completed the present invention.
Accordingly, an aspect of the present invention is directed to providing a pharmaceutical composition containing periodontal tissue-derived mesenchymal stem cells for prevention or treatment of male infertility.
Another aspect of the present invention is directed to providing a stem cell therapeutic agent containing periodontal tissue-derived mesenchymal stem cells for treatment of male infertility.
In order to achieve the above purpose, the present invention provides a pharmaceutical composition containing periodontal tissue-derived mesenchymal stem cells for prevention or treatment of male infertility.
In addition, the present invention provides a stem cell therapeutic agent containing periodontal tissue-derived mesenchymal stem cells for treatment of male infertility.
In addition, the present invention provides a method for treatment of male infertility, in which the method includes treating a subject with periodontal tissue-derived mesenchymal stem cells.
The pharmaceutical composition for prevention or treatment of male infertility according to the present invention recovers sex hormone levels in seminiferous tubules of atrophic testes and blood to normal levels through improvement in the activity of lipid metabolism enzymes in the liver, thereby exhibiting remarkable prophylactic and therapeutic effects on male infertility. In addition, the periodontal tissue-derived mesenchymal stem cells which are an active ingredient in the present invention can be obtained in the most non-invasive manner and as such, are expected to be advantageously used as a cell therapeutic agent for male infertility.
The present invention provides a pharmaceutical composition containing periodontal tissue-derived mesenchymal stem cells for prevention or treatment of male infertility.
Hereinafter, the present invention will be described in detail.
Terms not otherwise defined herein have the meanings commonly used in the technical field to which the present invention pertains.
As used herein, the term “stem cells” is a generic term for undifferentiated cells before differentiation into respective cells constituting tissue, and the stem cells have an ability to be differentiated into particular cells by particular differentiation stimulations (environment).
As used herein, the term “mesenchymal stem cells (MSCs)” is also referred to as mesenchymal stem cells or adult stem cells, and commonly refers to stem cells isolated and cultured from tissues other than embryos. Mesenchymal stem cells are present in small amounts in most tissues that have already been differentiated, and their presence has been identified in almost all tissues studied so far, such as umbilical cord blood, umbilical cord, teeth, eyes, placenta, hair follicles, lungs, and liver.
As used herein, the term “periodontal tissue-derived mesenchymal stem cells” includes both adult stem cells derived from teeth and periodontal tissue and cells similar in morphology to mesenchymal stem cells (mesenchymal stem cell-like cells). The periodontal tissue-derived mesenchymal stem cells have the advantage of minimizing tissue damage and being easily obtainable (non-invasively) compared to other adult stem cells present in the bone marrow or umbilical cord, and thus were used as an active ingredient of the present invention. The periodontal tissue is not limited thereto, but may be pulp tissue of primary or permanent teeth, periodontal ligament tissue, dental follicle tissue of erupting teeth, dental germ stem tissue, and root papillary tissue of immature permanent teeth, preferably wisdom teeth surrounding tissue.
In addition, the tissue may contain mesenchymal stem cells, in which the mesenchymal stem cells may be one or more selected from the group consisting of dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), and stem cells from apical papilla (SCAP), but are not limited thereto.
Such periodontal tissue-derived mesenchymal stem cells have been clinically applied to regenerative medicine to treat or improve peri-implantitis, nerve damage, and loss of gingival bone. However, so far, there have been no reports of any medical effects on male infertility.
In the present invention, the periodontal tissue is isolated from the human body and vitrified, and the vitrification method may use a method described in Korean Patent No. 10-1551900 (Patent Document 1), which is a related art of the present inventors.
In the present invention, as a method for isolating mesenchymal stem cells, methods known in the pertinent art may be used without limitation. For example, the mesenchymal stem cells may be isolated and purified from periodontal tissue, and the isolated mesenchymal stem cells may be cultured if necessary.
In the present invention, the male infertility is not limited thereto, but may be caused by obesity, diabetes, smoking, stress and non-alcoholic fatty liver disease, preferably obesity or non-alcoholic fatty liver disease (NAFLD).
In the present invention, the “pharmaceutical composition containing periodontal tissue-derived mesenchymal stem cells for prevention or treatment of male infertility” may be periodontal tissue-derived mesenchymal stem cells themselves, and include a composition containing a culture medium, and a substance generally used in the pertinent art when culturing stem cells, in addition to periodontal tissue-derived mesenchymal stem cells. In addition, it is meant to include a use of a substance secreted by periodontal tissue-derived mesenchymal stem cells or a substance extracted from periodontal tissue-derived mesenchymal stem cells in place of periodontal tissue-derived mesenchymal stem cells.
In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier, in addition to periodontal tissue-derived mesenchymal stem cells. For injection, the formulation may include a preservative, an analgesic, a solubilizer, a stabilizer or the like, and for topical administration, the formulation may include a base, an excipient, a lubricant, a preservative or the like.
A pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is typically used in the formulation. Examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, aromatics, emulsifiers, suspensions, and preservatives besides the above components.
The pharmaceutical composition of the present invention may be prepared in a unit dosage form or enclosed in a multi-dose container using a pharmaceutically acceptable carrier and/or excipient according to a method easily carried out by a skilled having ordinary skill in the technical field to which the present invention pertains. Herein, the formulation may be a solution in oil or an aqueous medium, suspension, or emulsion, and may further include a dispersing agent or a stabilizing agent.
The pharmaceutical composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically). The composition for parenteral administration (for example, injectable solution) according to the present invention may be injected in vivo by dispersion and/or dissolution thereof in a pharmaceutically acceptable carrier, for example, sterile purified water, a buffer of about pH 7, or saline solution. The pharmaceutical composition may include a conventional additive such as a preservative, a stabilizer or the like, if necessary.
In addition, in the present invention, the amount of mesenchymal stem cells may be injected in an amount of 104 to 1010 cells/injection, preferably 105 to 109 cells/injection, more preferably 106 to 108 cells/injection, and most preferably 5×107 to 108 cells/injection, but is not limited thereto. The dosage may be prescribed in various ways depending on factors such as a formulation method, an administration method, the age, weight, and pathosis of a patient, food, administration time, an administration route, an excretion rate, and sensitivity to reaction.
The pharmaceutical composition of the present invention may be administered one or more times, preferably administered three or more times at an interval of 1 to 3 weeks, and most preferably administered three or more times at an interval of 2 weeks.
In the present invention, the pharmaceutical composition may improve atrophy of seminiferous tubules in a testicle, and may improve sex hormone imbalance due to liver damage.
In example of the present invention, it was identified that when the periodontal tissue-derived mesenchymal stem cells were administered, the testicular structure of the male infertility animal model was recovered and the seminiferous tubules were improved, such as the formation of sperm in the lumen. It was identified that as the diameter length increased, the histological characteristics of the seminiferous tubules were remarkably recovered, and the number of Leydig cells producing testosterone significantly increased.
In addition, in another example of the present invention, it was identified that as the periodontal tissue-derived mesenchymal stem cells were administered, the testosterone/17β-estradiol value corresponding to the clinical indicator of the serum testosterone level and sex hormone balance increased, so that the sex hormone imbalance according to the high-fat diet (obesity) was significantly improved.
The pharmaceutical composition of the present invention may be used alone or in combination with methods for preventing or treating male infertility known in the pertinent art for the prevention and treatment of male infertility.
In addition, the present invention provides a stem cell therapeutic agent containing periodontal tissue-derived mesenchymal stem cells for treatment of male infertility.
In the present invention, the periodontal tissue is not limited thereto, but may be pulp tissue of primary or permanent teeth, periodontal ligament tissue, dental follicle tissue of erupting teeth, dental germ stem tissue, and root papillary tissue of immature permanent teeth, preferably wisdom teeth surrounding tissue.
As used herein, the term “cell therapeutic agent” refers to a drug used for the purpose of treatment, diagnosis and prevention, which contains a cell or tissue prepared through isolation from a human being, culture and specific operation (as provided by the US FDA), and refers to a drug used for the purpose of treatment, diagnosis and prevention through a series of behaviors of in vitro proliferating and sorting living autologous, allogenic and xenogenic cells or changing the biological characteristics of cells by other means for the purpose of recovering the functions of cells or tissues.
As used herein, the term “prevention” refers to all actions that inhibit male infertility or delay the progression of male infertility by administration of a pharmaceutical composition or a stem cell therapeutic agent according to the present invention.
As used herein, the term “treatment” refers to all actions that alleviate or beneficially change male infertility by administration of a pharmaceutical composition or a stem cell therapeutic agent according to the present invention.
In addition, the present invention provides a method for treatment of male infertility, in which the method includes treating a subject with periodontal tissue-derived mesenchymal stem cells.
The subject of the present invention may be a vertebrate, preferably a mammal, more preferably a rat, rabbit, guinea pig, hamster, dog, cat or human being, and most preferably a human being, but is not limited thereto.
The treatment of a subject with periodontal tissue-derived mesenchymal stem cells of the present invention may be a treatment performed one or more times, preferably a treatment performed three times or more at an interval of 1 to 3 weeks, and more preferably a treatment performed three or more times at an interval of 2 weeks.
Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to a person having ordinary skill in the pertinent art that the scope of the present invention is not limited by these examples without departing from the gist of the present invention. Statistical analysis was performed according to one-way ANOVA followed by Tukey's test using SPSS (SPSS Inc., Chicago, Ill., USA). Results are presented as mean±standard error. When P<0.05, it was determined to be significant.
All chemicals were purchased from SigmaAldrich® (St. Louis, Mo., USA), and the medium was purchased from Gibco Life Technologies (Gaithersburg, Md., USA). In order to obtain the periodontal tissue-derived mesenchymal stem cells, the present inventors established a new freezing protocol, and named it a vitrification method. The method is designed to effectively preserve cells in a tissue by using a composition for vitrification of tissue containing ethylene glycol, sucrose and glucose, and is described in Korean Patent No. 10-1551900 (Patent Document 1), which is a related art of the present inventors, which is incorporated herein by reference in its entirety.
More specifically, the periodontal tissue that was discarded after being extracted from an impacted wisdom teeth patient around the age of 20 was provided by the Department of Oral and Maxillofacial Surgery at Gyeongsang National University Hospital. The tooth tissue was separated from the extracted wisdom teeth using a sterile scalpel and vitrified, and was stored for at least 3 months and then thawed. After thawing the freezing tube stored in liquid nitrogen in the water at 37° C. for 1 to 2 minutes when thawing the tissue, DPBS containing 100 U/ml of penicillin/streptomycin and 0.25 μg/ml of amphotericin B (Invitrogen) was used to completely remove a cryopreservative, and was washed several times by centrifugation at 1500 rpm for 5 minutes. In order to isolate mesenchymal stem cells from the periodontal tissue, collagenase type IV (Sigma, USA) was enzymatically treated for 30 minutes. The treated tissue was filtered once each in a 100 μm cell strainer (BD Falcon™, USA) and 40 μm cell strainer (BD Falcon™ USA), and then transferred to a 15 ml conical tube (15 ml polypropylene conical tube; BD Falcon™, USA) and then washed twice by centrifugation at 1500 rpm for 5 minutes using DPBS containing penicillin/streptomycin and amphotericin B (Gibco, USA).
In 25 T-flasks (Nunc™, Roskilde, Denmark) using 4 mL of DMEM (Dulbecco's modified Eagle's Medium) containing 10% fetal bovine serum (FBS), 1% L-glutamine (Glutamax™ and 1% penicillin/streptomycin), the vitrified periodontal tissue-derived mesenchymal stem cells were cultured in 5% CO2, 37° C. and humid conditions. The medium was changed once every 3 days. Immediately before administration to the animals, 4 to 5 passages of cells were removed from the bottom of the culture container with 0.25% trypsin/EDTA and washed with DPBS. The isolated cells were counted using a hemocytometer, and 1×106 cells were diluted in 200 μl of cold DPBS and used for subsequent experiments (hereinafter referred to as MSCs).
Forty 8-week-old male C57BL/6J mice weighing 20-22 g were purchased from Central Lab Animal Inc. (Seoul, Korea) and used. All mice were bred at a room temperature of 25±2° C. and humidity of 30-40% by maintaining a 12-hour light-dark cycle, and allowed free access to water and food. Mice were randomly divided into 4 groups:
1. Control group (Con) (n=10): normal diet for 10 weeks
2. HFD group (n=10): high-fat diet containing 60 kcal % fat for 10 weeks+intraperitoneal administration of 200 μl of PBS after 10 weeks
3. MSCi group (n=10): high-fat diet containing 60 kcal % fat for 10 weeks+after 5, 7, and 9 weeks of a high-fat diet, intraperitoneal administration of 1×106/200 μl of MSCs each
4. MSCs group (n=10): high-fat diet containing 60 kcal % fat for 10 weeks+after 8 weeks of a high-fat diet, intraperitoneal administration of 1×106/200 μl of MSCs for 5 consecutive days.
All mice were weighed weekly, and after 10 weeks, 0.5 μL/g of tiletamine-zolazepam (Zoletil®) and 0.5 μL/g of xylazine (Rompun, Bayer Korea Ltd., Seoul, Korea) were injected and sacrificed. The mouse blood was collected by cardiac puncture, and the residual blood was removed by perfusion with PBS containing heparin. Liver and testicular tissues were obtained and weighed for tissue weight/body weight calculation. All animal experiments were performed according to the guidelines for the treatment and use of laboratory animals of Gyeongsang National University. The results are shown in
As shown in
In order to identify the therapeutic effect of periodontal tissue-derived mesenchymal stem cells on liver and testis, histological analysis was performed. Liver and testicular tissues were fixed in 4% formaldehyde, dehydrated, and then embedded in paraffin and cut into 5 μm thick sections. After removing the paraffin component from the tissue section using xylene, rehydration with ethanol was performed, and then staining with hematoxylin and eosin (H&E). After all the stained tissues were dehydrated, washed, and mounted with a permount (Fisher scientific, NH, USA), the degree of staining was checked with an optical microscope (Nikon Eclipse 80i) and Photo Imaging System (Canon 600D). Seminiferous tubule diameter and Leydig cell number were measured using NIS Elements BR3.2. A total of 150 seminiferous tubules were measured in 10 mice per group (3 fields per mouse, 5 seminiferous tubules randomly per field), and Leydig cells were counted in 30 captured images (3 fields per mouse, 10 mice). The results are shown in
As shown in
Through the above experimental results, it was identified that NAFLD and male infertility animal models in which testicular atrophy was induced by a high-fat diet were successfully prepared, and that the periodontal tissue-derived mesenchymal stem cells were effective in male infertility when administered. On the other hand, there was a slight difference in the treatment efficacy depending on the administration method. It was identified that at each time point after 5, 7, and 9 weeks of a high-fat diet, the MSCi group administered with mesenchymal stem cells showed a better therapeutic effect on body weight and seminiferous tubular recovery than the MSCs group administered with mesenchymal stem cells for 5 consecutive days after 8 weeks. In subsequent experiments, only the MSCi group was selected as the MSC group and the experiment was performed (hereinafter, referred to as the MSC group).
In order to evaluate apoptosis induced by a high-fat diet, TUNEL analysis was performed. The levels of apoptosis in liver and testicular tissues were evaluated using a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) kit (TUNEL, Roche, Ind., USA). Ten areas were randomly selected from each slide, and the number of brown-stained nuclei was counted as the number of TUNEL-positive cells under 400 magnifications, and the average of the measured numbers was calculated. All analyzes were performed blindly using NIS Elements BR3.2. The results are shown in
As shown in
In order to evaluate changes in liver function related to estrogen metabolism in an animal model of male infertility, the expression levels of aromatase and CYP1A2 were analyzed by immunohistochemistry. For immunohistochemistry (IHC), rehydrated sections were boiled in 0.1 M citric acid buffer (pH 6.0) for 10 minutes to restore antigen, and endogenous peroxidase activity was blocked by reacting with 3% H2O2 at room temperature for 30 minutes. Tissue sections were reacted with ABP (LS-B12124, LSBio), monoclonal mouse anti-cytochrome P450 1A2, CYP1A2 (sc-53241, Santacruz), and polyclonal rabbit anti-aromatase (ab18995, Abcam) diluted 1:100 overnight at 4° C. Thereafter, a secondary antibody IgG (Vector Laboratories, Burlingame, Calif., USA) diluted 1:200 and conjugated with biotin was applied, and then was reacted with ABC reagent (avidin-biotin-peroxidase complex; ABC Elite Kit, Vector Laboratories). All slides were color-developed with 0.05% diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich) and counterstained with Mayer's hematoxylin. Positive signals were analyzed in 30 fields per each group (3 random fields selected from 10 slides), and the ratio of positive signal area/all areas was calculated. The results are shown in
As shown in
Furthermore, as shown in
Mouse blood was collected by cardiac puncture by the method described in Example 2, and serum was collected by centrifugation at 400×G for 10 minutes and stored at −80° C. until use. In order to identify whether periodontal tissue-derived mesenchymal stem cells can improve the sex hormone imbalance induced by a high-fat diet and NALFD, testosterone in mouse serum (T; ADI-900-065, ENZO), 17(3-estradiol (E2; ADI-900-174, ENZO), FSH (LS-F39325, LSBio), LH (LSF22503, LSBio), TNF-α (ADI-900-047, ENZO), and IL-1β were analyzed by ELISA. The results are shown in
As shown in
Furthermore, as a result of measuring FSH and LH levels that stimulate the testes to produce ABP and testosterone to identify male hypogonadism, it was identified that both gonadotropins were significantly decreased in the HFD group, but increased significantly after MSC administration (D and E). Through the above results, it was identified that the decrease in ABP production was affected by the decrease in FSH caused by sex hormone imbalance.
The mechanism of action (Mode of Action, MoA) for treatment of male infertility of the periodontal tissue-derived mesenchymal stem cells of the present invention identified in the above Examples is shown in
Among the various animal models of inducing NAFLD, it is reported that the high-fat diet animal model has the pathological characteristics most similar to that of human NAFLD, and exhibits similar physiological changes such as weight gain, increased blood sugar, and increased metabolic disease.
The present inventors prepared an animal model induced by a high-fat diet in order to develop a new therapeutic agent for male infertility that can appear in men with obesity or NAFLD, and identified that when administered to the animal model with periodontal tissue-derived mesenchymal stem cells, the histological characteristics of the seminiferous tubules were restored, sex hormone imbalance was alleviated, various hormone metabolizing enzymes was expressed, and histological findings were normalized through fat metabolism and hepatocyte recovery.
Moreover, the periodontal tissue-derived mesenchymal stem cells which are an active ingredient in the present invention may be obtained in the most non-invasive manner and as such, are expected to be advantageously used as a cell therapeutic agent for male infertility.
From the foregoing, the specific portions of the present invention have been described in detail. Therefore, it is apparent to a person having ordinary skill in the pertinent art that such specific technology is merely a preferable embodiment, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0053424 | May 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/006091 | 5/8/2020 | WO | 00 |
