Medicine for repairing ovarian function

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for repairing an ovarian function and application thereof. The invention finds that NMDPEF (also known as S29434) can repair the ovarian function of a psychological stress individual, prevent premature activation of primordial follicles induced by psychological stress, improve the quality of oocytes and improve the fertilization ability and embryonic development potential of the oocytes. The NMDPEF protects a primordial follicle library of the psychological stress mouse and preserves fertility; mDPEF can improve the quality of the oocyte of the psychological stress mouse; the NMDPEF can improve the fertilization ability and blastocyst development rate of the oocyte of the psychological stress mouse. The medicine is easy to obtain, low in cost and suitable for large-scale production, and the treatment difficulty of patients is reduced; and the medicine has the advantages of obvious effect, quick response, shortened medication cycle, reduced toxic and side effects and improved patient compliance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug for repairing ovarian function and its application. Background Technology

[0002] Diminished ovarian reserve (DOR) refers to a decrease in the number of follicles and the quality of oocytes in the ovaries, leading to insufficient ovarian function and decreased fertility. It is accompanied by decreased levels of anti-Müllerian hormone (AMH), a decreased antral follicle count (AFC), and increased basal follicle-stimulating hormone (FSH) levels (Practice Committee of the American Society for Reproductive Medicine; Practice Committee of the American Society for Reproductive Medicine. Testing and interpreting measures of ovarian reserve: a committee opinion. Fertil Steril. 2020; 114(6): 1151-1157). Clinically, it mainly manifests as menstrual disorders, ovulation abnormalities, and infertility. Currently, there is no unified standard for determining DOR in clinical medicine. Furthermore, due to differences in the interpretation of the same test items by different testing institutions, it is difficult to determine its pathogenesis. At present, age, genetic factors, lifestyle, radiotherapy / chemotherapy, surgery and other factors are mainly identified as major risk factors (Ge Renjie, Hu Rongkui, Chen Hao, et al. Progress in the diagnosis and treatment of ovarian reserve decline with traditional Chinese medicine [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 35(1): 322-325.).

[0003] Stress refers to the comprehensive response of the body when exposed to stressors (factors that cause physical and mental tension in the body) (Boudarene M, Legros JJ, Timsit-Berthier M. Study of the stress response: role of anxiety, cortisol and DHEAs. Encephale. 2002; 28(2): 139-46). Psychological stress is closely related to human diseases. When psychological stress occurs, the body will undergo a comprehensive response involving the nervous, endocrine, and immune systems. Furthermore, a person's cognition of stress will further affect the body's response. When the body is in a strongly stimulating environment, the cerebral cortex, after comprehensive analysis, will cause a series of non-specific responses such as nerve excitation, increased hormone secretion, increased blood pressure, and increased heart rate. Extensive surveys have found a direct correlation between low fertility and anxiety. Compared to women with normal fertility, those who knew they would have difficulty conceiving or carrying a pregnancy to full term exhibited more pronounced anxiety (King RB. Subfecundity and anxiety in a nationally representative sample. Soc Sci Med. 2003; 56(4): 739-751). Furthermore, it was found that women with low fertility had a stronger desire to have their own children than women with normal fertility, but lower fertility further exacerbated their anxiety.

[0004] A single severe or prolonged psychological stress can affect human physical and mental health and may also have adverse effects on the reproductive endocrine system, leading to a variety of fertility-related diseases, including dysfunctional uterine bleeding, infertility, amenorrhea, and low libido (Cheng Xiang, Chen Zhengqiong, Xie Rongkai, et al. Clinical observation on the treatment of menopausal depression with fluoxetine hydrochloride combined with estrogen. Chongqing Medical Journal, 2005, 34 (5): 721-722; Lu Li, Li Jianping, Xue Yunzhen, et al. Relationship between emotional reaction, personality traits and menstrual disorders. Chinese Journal of Mental Health, 2005, 19 (3): 156-158). Psychological stress can affect follicle development and survival, ultimately leading to a decline in ovarian function (Zhai QY, Wang JJ, Tian Y, Liu X, Song Z. Review of psychological stress on oocyte and early embryonic development in female mice. Reprod Biol Endocrinol. 2020; 18(1): 101). The ovary produces eggs and secretes hormones. The development of follicles in the ovary includes primordial follicles, preantral follicles, antral follicles, and mature follicles. Preantral follicles and antral follicles are collectively referred to as growing follicles (Shah JS, Sabouni R, CaytonVaught KC, et al. Biomechanics and mechanical signaling in the ovary: a systematic review. J Assist Reprod Genet. 2018; 35(7): 1135-1148; Gruhn JR, Zielinska AP, Shukla V, et al. Chromosome errors in human eggs shape naturalfertility over reproductive life span. Science. 2019; 365(6460): 1466-1469).Chronic psychological stress can accelerate the activation of primordial follicles and disrupt the development of growing follicles, damaging oocyte quality and leading to premature depletion of ovarian reserve (Xu YP, Fu JC, Hong ZL, et al. Psychological stressors involved in the pathogenesis of premature ovarian insufficiency and potential intervention measures. Gynecol Endocrinol. 2024; 40(1): 2360085; Bleil ME, Adler NE, Pasch LA, et al. Depressive symptomatology, psychological stress, and ovarian reserve: a role for psychological factors in ovarian aging? Menopause. 2012; 19(11): 1176-1185). In addition, psychological stress can also affect the ovulation process. Persistent anxiety and psychological stress can disrupt the cascade of signaling molecules such as dopamine, serotonin, gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone, leading to ovulation abnormalities and infertility in individuals (Levine S, Muneyyirci-Delale O. Stress-Induced Hyperprolactinemia: Pathophysiology and clinical approach. Obstet Gynecol Int. 2018; 2018: 9253083).

[0005] The impact of psychological stress on ovarian function is gradually gaining attention from both doctors and patients, but timely and effective intervention methods are still lacking. Therefore, there is an urgent need for an effective and feasible intervention target and clinical strategy to protect and improve ovarian function in individuals experiencing psychological stress. Summary of the Invention

[0006] This invention discovers that NMDPEF (also known as S29434) can repair ovarian function, primarily by protecting the primordial follicle pool in the ovary after psychological stress, improving oocyte quality, enhancing oocyte fertilization capacity, and increasing the blastocyst development rate after fertilization. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides the application of NMDPEF in the preparation of drugs for repairing ovarian function.

[0008] Furthermore, the ovarian function mentioned refers to the ovarian function of individuals under stress.

[0009] Furthermore, the stressed individuals are those experiencing psychological stress.

[0010] Furthermore, the NMDPEF includes pharmaceutically acceptable salts, solvent compounds, and derivatives.

[0011] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0012] Furthermore, the drug can be formulated into various dosage forms, including tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, and suppositories.

[0013] Furthermore, the repair of ovarian function includes protecting the primordial follicle bank in the ovary, improving oocyte quality, enhancing the fertilization capacity of oocytes, and increasing the blastocyst development rate after oocyte fertilization.

[0014] Furthermore, the NMDPEF dosage is selected from 0.1-100 mg / kg / d, preferably 4.5 mg / kg / d.

[0015] In a second aspect, the present invention provides a pharmaceutical composition comprising NMDPEF and another drug for repairing ovarian function, the pharmaceutical composition having at least one of the following effects: 1) Protect the primordial follicle pool in the ovary; 2) Improve oocyte quality; 3) Enhance the fertilization capacity of oocytes; 4) Improve the blastocyst development rate after oocyte fertilization.

[0016] Furthermore, the ovarian function mentioned refers to the ovarian function of individuals under stress.

[0017] Furthermore, the stressed individuals are those experiencing psychological stress.

[0018] Furthermore, NMDPEF in the pharmaceutical composition can be used alone or in combination with other drugs for repairing ovarian function.

[0019] Furthermore, the NMDPEF includes pharmaceutically acceptable salts, solvent compounds, and derivatives.

[0020] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0021] Furthermore, the drug can be formulated into various dosage forms, including tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, and suppositories.

[0022] Furthermore, the NMDPEF dosage is selected from 0.1-100 mg / kg / d, preferably 4.5 mg / kg / d.

[0023] Thirdly, the present invention provides the application of NMDPEF in repairing ovarian function, wherein the NMDPEF functions in the following manner: 1) Protect the primordial follicle pool in the ovary; 2) Improve oocyte quality; 3) Enhance the fertilization capacity of oocytes; 4) Improve the blastocyst development rate after oocyte fertilization.

[0024] Furthermore, the ovarian function mentioned refers to the ovarian function of individuals under stress.

[0025] Furthermore, the stressed individuals are those experiencing psychological stress.

[0026] Furthermore, the NMDPEF includes pharmaceutically acceptable salts, solvent compounds, and derivatives.

[0027] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0028] Furthermore, the drug can be formulated into various dosage forms, including tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, and suppositories.

[0029] Furthermore, the NMDPEF dosage is selected from 0.1-100 mg / kg / d, preferably 4.5 mg / kg / d.

[0030] Beneficial effects This invention discovers that NMDPEF has the effect of repairing ovarian function in individuals with psychological stress, can intervene in the trend of ovarian function decline in individuals with psychological stress, and improve fertility, with the following advantages: 1. Compared with the untreated control group, the number of primordial follicles decreased in the tail suppression (TS) group. This trend was effectively reversed in the NMDPEF rescue group, meaning that NMDPEF application can protect the primordial follicle pool of psychologically stressed mice and preserve fertility. 2. Compared with the untreated control group, the oocyte mortality rate and fragmentation rate increased after superovulation in mice in the tail suspension stress group. This degenerative change was effectively reversed in the NMDPEF rescue group. The morphology and quality of the oocytes obtained after superovulation were significantly improved, that is, NMDPEF can improve the quality of oocytes in psychologically stressed mice. 3. Compared with the untreated control group, the 2-cell rate and blastocyst rate of oocytes in the tail suspension stress group were significantly reduced after fertilization. This trend was significantly reversed in the NMDPEF rescue group, that is, NMDPEF improves the fertilization capacity and blastocyst development rate of oocytes in psychologically stressed mice. 4. The drug is easy to obtain, low in cost, and can be mass-produced, reducing the difficulty of treatment for patients; 5. This drug has significant effects, works quickly, shortens the treatment cycle, reduces drug toxicity and side effects, and improves patient compliance. Attached Figure Description

[0031] Figure 1 Applying NMDPEF can protect the primordial follicle bank in the ovaries of psychologically stressed mice. 1A HE staining image of ovarian tissue section; 1B， Statistical analysis revealed differences in serum anti-Müllerian hormone (AMH) levels and the number of primordial follicles in the ovaries among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the stress group showed a significant increase in AMH, a significant decrease in the number of primordial follicles in the ovaries, and a significant increase in the ratio of growing follicles to primordial follicles. These trends were significantly alleviated after NMDPEF administration.

[0032] Figure 2 Applying NMDPEF improves the survival and quality of oocytes in stressed mice. 2A The morphology of oocytes collected from stressed mice after superovulation treatment. The small arrows indicate dead cells, and the arrowheads indicate fragmented cells. 2B Statistical analysis revealed differences in oocyte mortality (a) and fragmentation rate (b) among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the oocyte mortality and fragmentation rates were significantly increased in the stress group, but the application of NMDPEF significantly reversed these trends, bringing them close to the control group levels.

[0033] Figure 3 Applying NMDPEF improves the fertilization and blastocyst development potential of oocytes after stress. 3A The morphology of oocytes after fertilization, developing into the 2-cell stage and the blastocyst stage; 3B Statistical analysis revealed differences in 2-cell rate and blastocyst development rate among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the 2-cell rate and blastocyst rate were significantly decreased in the stress group. The application of NMDPEF significantly reversed this trend, bringing them close to the control group levels. Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0036] Terminology Explanation NMDPEF (also known as S29434), its chemical formula is C0 21 H 18 N4O3, with the structural formula shown in Formula I, is an effective, competitive, selective, and cell membrane-penetrating inhibitor of quinone oxidoreductase 2 [N-ribosyldihydronicotinamide(NRH):quinone oxidoreductase 2, NQO2].

[0037] (Formula I) Example 1: NMDPEF Concentration Screening The method described in the references (The antidote effect of quinone oxidoreductase 2 inhibitor against paraquat-induced toxicity in vitro and in vivo) Br JPharmacol (2013; 168: 46-59. doi: 10.1111 / j.1476-5381.2012.01870.x), after designing different concentrations, it was found that the optimal dosage of NMDPEF was 4.5 mg / kg / d, and this concentration was used in subsequent examples.

[0038] Example 2: NMDPEF protects the primordial follicle bank in psychologically stressed mice and preserves fertility. A. Test reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline and NMDPEF powder.

[0039] Preparation of NMDPEF stock solution and working solution: Dissolve NMDPEF in DMSO to prepare a 9 mg / ml stock solution, and aliquot and store at 20 °C. Before use, dilute the stock solution with 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline to prepare a 0.9 mg / ml working solution.

[0040] B. Test methods 1) Experimental mice were divided into three groups: control group (Ctrl), tail suspension stress group (TS), and NMDPEF rescue group (TS + NMDPEF). Each group consisted of five 2-month-old female mice with a C57BL / 6J background, weighing 20 grams each. Mice in the stress and NMDPEF rescue groups underwent tail suspension for 10 minutes daily for 10 consecutive days. The rescue group received an intraperitoneal injection of 100 ml of NMDPEF working solution two hours before each tail suspension. The stress group received an equal volume of the solvent (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline). The control group received no treatment. 2) The actual dose of NMDPEF administered intraperitoneally to mice was 4.5 mg / kg / day; 3) After 10 consecutive days of treatment, the ovaries of mice were removed, fixed and embedded, and sectioned for follicle counting.

[0041] C. Test results Figure 1 A The image shown is a HE staining image of an ovarian tissue section. Figure 1 B As shown, statistical analysis revealed differences in serum anti-Müllerian hormone (AMH) levels and the number of primordial follicles in the ovaries among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the AMH level in the stress group was significantly higher ( P <0.01, applying NMDPEF significantly reversed the upward trend of AMH ( P <0.01, close to the control group level. Compared with the control group, the number of primordial follicles in the ovaries of the stress group was significantly reduced ( P <0.01, the ratio of growing follicles to primordial follicles increased significantly ( P <0.01, the decreasing trend of the number of primordial follicles was significantly alleviated after NMDPEF application, and the ratio of growing follicles to primordial follicles decreased significantly and approached the level of the control group ( P <0.01). The above data indicate that ovarian reserve is significantly reduced in individuals experiencing psychological stress, and that applying NMDPEF can reverse this trend and protect the ovaries.

[0042] Example 3: NMDPEF improves oocyte quality in psychologically stressed mice A. Test reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline and NMDPEF powder.

[0043] Preparation of NMDPEF stock solution and working solution: Dissolve NMDPEF in DMSO to prepare a 9 mg / ml stock solution, and aliquot and store at 20 °C. Before use, dilute the stock solution with 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline to prepare a 0.9 mg / ml working solution.

[0044] B. Test methods 1) Experimental mice were divided into three groups: control group (Ctrl), tail suspension stress group (TS), and NMDPEF rescue group (TS + NMDPEF). Each group consisted of five 2-month-old female mice with a C57BL / 6J background, weighing 20 grams each. Mice in the stress and NMDPEF rescue groups underwent tail suspension for 10 minutes daily for 10 consecutive days. The rescue group received an intraperitoneal injection of 100 ml of NMDPEF working solution two hours before each tail suspension. The stress group received an equal volume of the solvent (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline). The control group received no treatment. 2) The actual dose of NMDPEF administered intraperitoneally to mice was 4.5 mg / kg / day; 3) Ten days after treatment, mice were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) 48 hours later. Oocytes were then collected from the ampulla of the mouse oviduct 14 hours later for morphological examination and counting.

[0045] C. Test results Figure 2 A As shown, the morphology of oocytes is illustrated, with small arrows indicating dead cells and arrowheads indicating fragmented cells. Figure 2 B As shown, statistical analysis revealed differences in oocyte mortality and fragmentation rate among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the oocyte mortality rate in the stress group was significantly higher.P <0.05) and fragmentation rate ( P <0.01) significantly increased mortality, and the mortality rate was significantly reversed after NMDPEF administration ( P <0.05) and the upward trend of fragmentation rate ( P The value was <0.01, close to the control group level. These data suggest that NMDPEF can significantly improve the survival and quality of oocytes in psychologically stressed mice.

[0046] Example 4: NMDPEF improves the fertilization capacity and blastocyst development rate of oocytes in psychologically stressed mice. A. Test reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline and NMDPEF powder.

[0047] Preparation of NMDPEF stock solution and working solution: Dissolve NMDPEF in DMSO to prepare a 9 mg / ml stock solution, and aliquot and store at 20 °C. Before use, dilute the stock solution with 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline to prepare a 0.9 mg / ml working solution.

[0048] B. Test methods 1) Experimental mice were divided into three groups: control group (Ctrl), tail suspension stress group (TS), and NMDPEF rescue group (TS + NMDPEF). Each group consisted of five 2-month-old female mice with a C57BL / 6J background, weighing 20 grams each. Mice in the stress and NMDPEF rescue groups underwent tail suspension for 10 minutes daily for 10 consecutive days. The rescue group received an intraperitoneal injection of 100 ml of NMDPEF working solution two hours before each tail suspension. The stress group received an equal volume of the solvent (10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline). The control group received no treatment. 2) The actual dose of NMDPEF administered intraperitoneally to mice was 4.5 mg / kg / day; 3) Ten days after treatment, mice were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG), followed by 10 IU of human chorionic gonadotropin (hCG) 48 hours later. Oocytes were then collected from the ampulla of the fallopian tubes of the mice 14 hours later. 4) The collected oocytes were subjected to in vitro fertilization experiments. The number and ratio of 2-cells and blastocysts were counted at 24 and 96 hours after the fertilization experiment to assess the fertilization capacity of the oocytes and the developmental potential of the embryo.

[0049] C. Test results Figure 3 Figure A shows the morphology of an oocyte after fertilization, developing into the 2-cell stage and the blastocyst stage. Figure 3 As shown in Figure B, statistical analysis revealed differences in 2-cell rate and blastocyst development rate among the control group (Ctrl), the tail suspension stress group (TS), and the NMDPEF rescue group (TS + NMDPEF). Compared to the control group, the stress group showed a significant decrease in both 2-cell rate and blastocyst development rate. P <0.05, applying NMDPEF significantly reversed the above trend ( P <0.05), close to the control group level. These data suggest that NMDPEF can improve the fertilization capacity and blastocyst developmental potential of oocytes in psychologically stressed mice.

Claims

1. Application of NMDPEF in the preparation of drugs for repairing ovarian function.

2. The application as described in claim 1, wherein the ovarian function is the ovarian function of a stressed individual; and the stressed individual is a psychologically stressed individual.

3. The application as described in claim 1, wherein the NMDPEF comprises pharmaceutically acceptable salts, solvent compounds, and derivatives thereof; and the dose of the NMDPEF is 4.5 mg / kg / day.

4. The application as described in claim 1, wherein the repair of ovarian function includes protecting the primordial follicle bank in the ovary, improving oocyte quality, enhancing the fertilization capacity of oocytes, and increasing the blastocyst development rate after oocyte fertilization.

5. In the application described in claim 1, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

6. A pharmaceutical composition comprising NMDPEF and another drug for restoring ovarian function, the pharmaceutical composition having at least one of the following effects: 1) Protect the primordial follicle pool in the ovary; 2) Improve oocyte quality; 3) Enhance the fertilization capacity of oocytes; 4) Improve the blastocyst development rate after oocyte fertilization.

7. The pharmaceutical composition of claim 6, wherein the ovarian function is the ovarian function of a stressed individual; the stressed individual is a psychologically stressed individual.

8. The pharmaceutical composition of claim 6, wherein the NMDPEF comprises pharmaceutically acceptable salts, solvent compounds, and derivatives thereof; and the dose of the NMDPEF is 4.5 mg / kg / day.

9. The pharmaceutical composition of claim 6, wherein one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition; the pharmaceutical composition may be formulated into various dosage forms, including tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules and suppositories.

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