Application of NMDPEF in improvement of ovarian function of elderly individuals

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for improving ovarian functions. By detecting the influence of the NMDPEF on primordial follicle reserve of an advanced ovary, ovary ovulation rate, oocyte quality and ovary microenvironment, it is found that after NMDPEF treatment, the trend that the number of follicles in the ovary is decreased is obviously relieved, the form and number of oocytes harvested by serum AMH recovery and superovulation are remarkably improved, iron deposition in the ovary is reduced, and the ovary ovulation rate is reduced. The oxidative stress index, the DNA damage index, the aging index and the iron metabolism index are all recovered. Therefore, the NMDPEF can improve the microenvironment of the elderly ovary, delay the depletion of the primordial follicle reservoir, increase the ovulation rate and increase the quality of oocytes.

Description

Technical Field

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

[0002] The ovary is a vital organ responsible for reproduction. It periodically produces and releases mature eggs, which is a prerequisite for female conception. At the same time, as a gonadal organ, the ovary can secrete a variety of sex hormones, including estrogen (Ez) and anti-Müllerian hormone (AMH), which play an important role in maintaining female reproductive health, promoting the development of female secondary sexual characteristics, and regulating the menstrual cycle (Wood CL, Lane LC, Cheetham T. Puberty: Normal physiology (brief overview) [J]. Best Pract Res Clin Endocrinol Metab. 2019 Jun;33(3):101265.). Women are born with a fixed number of oocytes. With age, the number and quality of oocytes in the ovaries decrease, leading to a progressive decline in ovarian function—a process known as ovarian aging (Wang X, Wang L, Xiang W. Mechanisms of ovarian aging in women: areview [J]. J Ovarian Res. 2023 Apr 6;16(1):67.). Clinically, ovarian quality is assessed using sex hormone levels (follicle-stimulating hormone (FSH), E2, AMH) and antral follicle count. Hormone imbalances caused by ovarian aging not only affect a woman's fertility but also disrupt her metabolic processes, increasing the risk of serious illnesses. A study statistically analyzed the risk association between menopause and cardiovascular disease, and the results showed that ovarian aging increases the risk factors for cardiovascular disease (high levels of triglycerides, high-density lipoprotein, glucose, waist circumference and blood pressure).

[0003] Female aging begins with ovarian aging, which in turn begins with a decline in ovarian reserve (OR). Ovarian reserve is comprised of follicles at various stages within the ovary, and it gradually depletes with age. This process is primarily driven by the physiological structure of the ovary, as the most important pool of follicles constituting the ovarian reserve is determined during the embryonic stage (Zhu Q, LiY, Ma J, Ma H, Liang X. Potential factors result in diminished ovarian reserve: a comprehensive review [J]. J Ovarian Res. 2023 Oct 25;16(1):208.). Therefore, without an increase in the number of eggs, cyclical ovulation is an inevitable consequence of declining ovarian reserve, manifested as a reduction in the number of primordial follicles. Meanwhile, the number of primordial follicles is affected by the balance between dormancy and activation. The environment surrounding the primordial follicles is called the extracellular matrix (ECM). Elastin and collagen in the ECM play a role in maintaining the dormant state of primordial follicles (Nagamatsu G, Shimamoto S, Hamazaki N, et al. Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes [J]. Sci Adv. 2019 Jun 26;5(6):eaav9960.). In the ovaries of menopausal women, the content of elastin and collagen in the ECM is significantly reduced. This indicates that with age, the protective effect of the ovary on primordial follicles weakens, leading to the loss of primordial follicles. As is well known, the process of primordial follicles developing into growing follicles (growing follicles are a collective term for primary and secondary follicles) requires a rich vascular network on the follicle surface to provide nutrition (Xu X, Mu L, Li L, et al. Imaging and tracing the pattern of adult ovarian angiogenesis impies a strategy against female reproductive aging [J]. Sci Adv. 2022 Jan14;8(2):eabi8683.). However, the ovarian angiogenesis capacity declines with age.Single-cell sequencing of ovarian cells from 9-month-old mice revealed decreased expression of angiogenesis-related endothelial growth factor (VEGF) and a significant reduction in the number of endothelial cells constituting blood vessels, indicating reduced blood vessel numbers and suppressed angiogenesis in aging ovaries (Isola JVV, Ocañas SR, Hubbard CR, Ko S, Mondal SA, Hense JD, Carter HNC, Schneider A, Kovats S, Alberola-Ila J, Freeman WM, Stout MB. A single-cell atlas of the aging mouseovary [J]. Nat Aging. 2024 Jan;4(1):145-162.). Furthermore, the ovarian inflammatory microenvironment is a crucial factor affecting follicular development and inducing follicular atresia, and aging exacerbates this inflammatory microenvironment. ScRNA-seg analysis of ovarian cells from aged mice showed that with increasing age, the number of pro-inflammatory immune cells in the ovary increased, along with increased transcription levels of pro-inflammatory factors IFN-γ and IL-17.

[0004] Hormone replacement therapy is a common treatment for hypohormia, which can alleviate the discomfort caused by the decline in hormone levels due to ovarian dysfunction. However, the long-term use of this method may lead to cancer and cardiovascular risks, and there are no effective preventive measures (Cho L, Kaunitz AM, Faubion SS, et al. Rethinking Menopausal Hormone Therapy: For Whom, What, When, and How Long? [J]. Circulation. 2023 Feb 14;147(7):597-610.). For women who want to have children, assisted reproductive technologies such as in vitro fertilization (IVF) provide the possibility of fertility. Although IVF can successfully solve infertility, it cannot reverse the effect of age on oocyte quality (Shang Y, Song N, He R, et al. Antioxidants and Fertility in Women with Ovarian Aging: A Systematic Review and Meta-Analysis. Adv Nutr. 2024 Aug;15(8):100273.). Therefore, existing strategies still have significant limitations. Most of them have unstable efficacy and are mostly symptomatic supportive, unable to effectively alleviate the declining trend of primordial follicle pool and oocyte quality. Developing more drugs to delay ovarian aging and prolong reproductive life is urgently needed.

[0005] NMDPEF (also known as S29434) is a molecular structure with the following structure: C 21 H 18 N4O3 compounds have been clearly identified in existing research as highly efficient, competitive, and cell-membrane-penetrating quinone reductase 2 (QR2) inhibitors, exhibiting significant activity in various biological systems. Research data show that NMDPEF has a significant inhibitory concentration (IC50) for human QR2. 50 The NMDPEF concentration reaches 5-16 nanomolar levels and exhibits significantly higher selectivity for QR2 than for QR1, demonstrating its excellent target specificity. At the cellular level, NMDPEF has been shown to effectively induce autophagy. These properties make NMDPEF an important chemical probe, widely used in scientific research to explore QR2-related signal transduction pathways, the molecular regulatory mechanisms of autophagy, and the role of oxidative stress in physiological and pathological processes. Currently, there are no studies on the effects of NMDPEF on ovarian function in elderly individuals. Summary of the Invention

[0006] This invention discovers that NMDPEF can improve the ovarian microenvironment in older women, delay the depletion of the primordial follicle bank, increase ovulation rate, and improve oocyte quality. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides the use of NMDPEF or pharmaceutically acceptable salts, solvent compounds, or derivatives thereof in the preparation of drugs for improving ovarian function in elderly individuals; wherein NMDPEF functions in the following manner: 1) NMDPEF improves the ovarian microenvironment in older women; 2) NMDPEF delays the depletion of the primordial follicle pool in older individuals; 3) NMDPEF improves ovulation rate in older individuals; 4) NMDPEF improves oocyte quality in older individuals.

[0008] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0009] Furthermore, the drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

[0010] In a second aspect, the present invention provides a pharmaceutical composition comprising NMDPEF and another drug for improving ovarian function in elderly individuals, the pharmaceutical composition having at least one of the following effects: 1) NMDPEF improves the ovarian microenvironment in older women; 2) NMDPEF delays the depletion of the primordial follicle pool in older individuals; 3) NMDPEF improves ovulation rate in older individuals; 4) NMDPEF improves oocyte quality in older individuals.

[0011] Furthermore, NMDPEF in the pharmaceutical composition can be used alone or in combination with other drugs that improve ovarian function in elderly individuals.

[0012] Furthermore, the other drugs for improving ovarian function in older individuals include, but are not limited to, hormonal drugs and nutritional antioxidants; the hormonal drugs include estradiol valerate tablets, dydrogesterone tablets, or dehydroepiandrosterone; the nutritional antioxidants include coenzyme Q10, N-acetylcysteine, and vitamins D and E.

[0013] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0014] Furthermore, the drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

[0015] Beneficial effects 1. This invention found that after NMDPEF treatment, the decline in the number of follicles in the ovaries of elderly individuals was significantly slowed down, and serum AMH levels rebounded; indicating that NMDPEF can delay the depletion of primordial follicles in elderly individuals and preserve fertility.

[0016] 2. This invention found that after NMDPEF treatment, the morphology and quantity of oocytes collected from superovulation in older individuals were significantly improved; indicating that NMDPEF can increase the ovulation rate and improve the quality of oocytes in older individuals.

[0017] 3. This invention found that after NMDPEF treatment, iron deposition in the ovaries of elderly individuals decreased, and oxidative stress indicators, DNA damage indicators, aging indicators, and iron metabolism indicators all recovered; indicating that NMDPEF can improve the ovarian microenvironment of elderly individuals.

[0018] These results suggest that NMDPEF is involved in improving oxidative stress levels in the ovarian microenvironment of older mice, regulating follicle development and survival. NMDPEF can improve the ovarian microenvironment in older mice, delay the depletion of the primordial follicle pool, increase ovulation rate, and improve oocyte quality. Attached Figure Description

[0019] Figure 1NMDPEF was used to improve the number and quality of ovulation in older mice. The main results were: after injection of NMDPEF3 (4.5 mg / kg qd) into older mice (10-12 months old), the number of primordial follicles was significantly higher than that in the older control group (A, B), and serum AMH levels rose (C).

[0020] Figure 2 NMDPEF was used to improve the quantity and quality of superovulation in older mice. The main results were: older mice (10-12 months old) treated with NMDPEF3 (4.5 mg / kg qd) showed a significant increase in the number of mature oocytes harvested after hormonal superovulation (A, B) and improved oocyte quality (C, D).

[0021] Figure 3 NMDPEF can improve the microenvironment of aging ovaries, mainly as follows: after injection of NMDPEF (4.5 mg / kg .qd) into aged mice (10-12 months old), iron deposition in the ovaries decreased (A), and oxidative stress indicators, DNA damage indicators, aging indicators and iron metabolism indicators were all restored (B). Detailed Implementation

[0022] 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.

[0023] 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.

[0024] Material description: NMDPEF (also known as S29434) has the molecular structural formula C 21 H 18 N4O3 is an effective, competitive, selective, cell membrane-permeable quinone reductase 2 (QR2) inhibitor.

[0025] Example 1: NMDPEF delays primordial follicle depletion and preserves fertility 1.1 Reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline, and NMDPEF.

[0026] 1.2 Preparation of NMDPEF working solution Dissolve NMDPEF in DMSO to prepare a concentrated stock solution of 13.5 mg / mL, aliquot and store. When using, prepare a working solution according to the ratio of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline. The specific concentration used is 1.35 mg / mL.

[0027] 1.3 Methods 1) The experimental mice were divided into three groups: a young control group, an old control group, and an old NMDPEF group, with 5 female mice of 2 / 12 / 12 months age with a C57BL / 6 background in each group.

[0028] 2) Take out the working reagents and prepare the working solution according to the ratio of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline before use; 3) The drug is administered via intraperitoneal injection at a dose of 4.5 mg / kg, once daily for 2 weeks; 4) Two weeks after drug treatment, the ovaries were removed, fixed and embedded, sectioned for follicle counting, and serum was collected for AMH content detection.

[0029] 1.4 Results Two weeks after drug treatment, follicle counting showed a rebound in the number of primordial follicles and the total number of follicles in the treatment group. Figure 1 B), serum AMH levels have rebounded ( Figure 1 C).

[0030] Example 2: NMDPEF can increase ovulation rate and improve oocyte quality. 1.1 Reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline, NMDPEF.

[0031] 1.2 Preparation of NMDPEF working solution Dissolve NMDPEF in DMSO to prepare a concentrated stock solution of 13.5 mg / mL, aliquot and store. When using, prepare a working solution according to the ratio of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline. The specific concentration used is 1.35 mg / mL.

[0032] 1.3 Methods 1) The experimental mice were divided into three groups: a young control group, an old control group, and an old NMDPEF group, with 5 female mice of 2 / 12 / 12 months age with a C57BL / 6 background in each group.

[0033] 2) Take out the working reagents and prepare the working solution according to the ratio of 10% DMSO, 40% PEG 300, 5% Tween 80 and 45% physiological saline before use; 3) The drug is administered via intraperitoneal injection at a dose of 4.5 mg / kg, once daily for 2 weeks; 4) Two weeks after drug treatment, injectable gonadotropin (10 IU) was administered, followed by injectable human chorionic gonadotropin (10 IU) 46 hours later. Oocytes from the ampulla of the mouse oviduct were collected 14 hours later for morphological examination and counting.

[0034] 1.4 Results Two weeks after drug treatment, the morphology and number of oocytes collected from superovulation were significantly improved. Figure 2 ).

[0035] Example 3: NMDPEF can improve the ovarian microenvironment in older women. 1.1 Reagents Dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), Tween 80, physiological saline, NMDPEF.

[0036] 1.2 Preparation of NMDPEF working solution Dissolve NMDPEF in DMSO to prepare a concentrated stock solution of 13.5 mg / mL, aliquot and store. When using, prepare a working solution according to the ratio of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline. The specific concentration used is 1.35 mg / mL.

[0037] 1.3 Methods 1) The experimental mice were divided into three groups: a young control group, an old control group, and an old NMDPEF group, with 5 female mice of 2 / 12 / 12 months age with a C57BL / 6 background in each group.

[0038] 2) Take out the working reagents and prepare the working solution according to the ratio of 10% DMSO, 40% PEG300, 5% Tween 80 and 45% physiological saline before use; 3) The drug is administered via intraperitoneal injection at a dose of 4.5 mg / kg, once daily for 2 weeks; 4) Two weeks after drug treatment, the ovaries were removed, fixed, embedded, and sectioned for Prussian blue staining; at the same time, the ovaries were removed for Western blot analysis to detect changes in ovarian DNA damage, oxidative stress indicators, and iron metabolism-related proteins.

[0039] 1.4 Results After two weeks of drug treatment, ovarian fibrosis and iron deposition were alleviated. Figure 3 A). Ovarian fibrosis markers, DNA damage, oxidative stress markers, and iron metabolism-related proteins all recovered. Figure 3 ).

Claims

1. The use of NMDPEF or its pharmaceutically acceptable salts, solvent compounds, or derivatives in the preparation of drugs for improving ovarian function in elderly individuals, characterized in that, The NMDPEF works in the following ways: 1) NMDPEF improves the ovarian microenvironment in older women; 2) NMDPEF delays the depletion of the primordial follicle pool in older individuals; 3) NMDPEF improves ovulation rate in older individuals; 4) NMDPEF improves oocyte quality in older individuals.

2. The application as described in claim 1, characterized in that, The drug may also contain one or more pharmaceutically acceptable carriers.

3. The application as described in claim 1, characterized in that, The drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises NMDPEF and another drug for improving ovarian function in older individuals, and the pharmaceutical composition has at least one of the following effects: 1) NMDPEF improves the ovarian microenvironment in older women; 2) NMDPEF delays the depletion of the primordial follicle pool in older individuals; 3) NMDPEF improves ovulation rate in older individuals; 4) NMDPEF improves oocyte quality in older individuals.

5. The pharmaceutical composition according to claim 4, characterized in that, NMDPEF in the pharmaceutical composition can be used alone or in combination with other drugs that improve ovarian function in older individuals.

6. The pharmaceutical composition according to claim 4, characterized in that, One or more pharmaceutically acceptable carriers may also be added to the pharmaceutical composition.

7. The pharmaceutical composition according to claim 4, characterized in that, The drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

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