Composition for improving ovarian function damage and degeneration and preparation method thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a pharmaceutical composition for improving ovarian function damage and degeneration and a preparation method thereof. The invention discloses the GnRH agonist effect of dihydromyricetin DMY for the first time, and the affinity of dihydromyricetin DMY to a GnRH receptor and the GnRH agonist effect of dihydromyricetin DMY are verified through in-vitro experiments. Model animal experiments further prove that the dihydromyricetin DMY has a protective effect on ovarian function impairment caused by chemotherapy. Experiments prove that the dihydromyricetin DMY is given while the cyclophosphamide (CTX) is used for chemotherapy, so that the damage of the dihydromyricetin DMY to the ovarian function during chemotherapy can be reduced, and a protection effect is also achieved on the recovery of the ovarian function after drug withdrawal. The protection effect is reflected on the endocrine function and the reserve function of the ovary, and the preserved ovary reserve function can achieve the pregnancy result after chemotherapy is finished, which indicates that the dihydromyricetin DMY also plays the protection effect on the fertility function of the ovary.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a composition for improving ovarian function damage and degeneration and a preparation method thereof. BACKGROUND

[0002] Advances in the field of tumor treatment have significantly improved patient survival rates, but the long-term complications that follow, especially the damage to female ovarian function caused by chemotherapy, are becoming a key issue affecting the quality of life of patients. Chemotherapeutic drugs, while killing rapidly proliferating tumor cells, cause irreversible damage to ovarian follicles that are also in an active state of division, thereby triggering premature ovarian insufficiency (POI), leading to loss of fertility, endocrine disorders, and a series of health problems.

[0003] The ovary, as the core organ of female reproductive and endocrine function, its reserve function depends on the number and quality of primordial follicles. The primordial follicle pool is fixed at birth and gradually depletes with age, and the intervention of chemotherapeutic drugs can accelerate this process, even leading to irreversible follicular depletion. The damage of chemotherapeutic drugs to the ovary is highly selective, and its core mechanism lies in the killing effect on rapidly dividing cells. The preantral follicle and antral follicle in the ovary are in an active growth phase, and are particularly sensitive to chemotherapeutic drugs, which can directly damage the DNA of oocytes, leading to follicular apoptosis or necrosis, such as alkylating agents (e.g. cyclophosphamide), platinum drugs, etc. Chemotherapeutic drugs can also damage the function of granulosa cells in the ovary, affecting their ability to secrete hormones and nutrients, and indirectly leading to follicular development arrest. Some drugs can reduce ovarian blood perfusion, reducing the nutritional supply and metabolic efficiency of follicles.

[0004] Unlike the damage to the gastrointestinal tract and bone marrow, the destruction of ovarian follicles is often irreversible—once the original follicle pool is depleted, the ovarian function cannot be restored by self-repair, which is the most prominent feature of chemotherapy-induced ovarian damage. The clinical manifestations of chemotherapy-induced ovarian damage are diverse, and as the degree of damage increases, short-term symptoms include perimenopausal symptoms such as menstrual disorders (such as cycle disorders, reduced menstrual flow), hot flashes, night sweats, insomnia, etc. during or shortly after chemotherapy; long-term effects, if the damage is severe, can lead to permanent amenorrhea and POI, manifested as elevated follicle-stimulating hormone (FSH) and decreased estrogen levels, which can lead to infertility, osteoporosis, increased risk of cardiovascular disease, and other problems; in terms of psychological burden, the loss of fertility has a significant impact on the psychology of young patients, research shows that about 60% of young female cancer patients list fertility needs as a core indicator of quality of life after treatment, and POI can lead to psychological barriers such as anxiety and depression. It is worth noting that the degree of damage is closely related to the type, dose, and course of chemotherapy drugs and the age of the patient. For example, alkylating agents have the strongest ovarian toxicity, and the incidence of POI in young women receiving cyclophosphamide treatment can reach 50%-80%; the older the patient (especially > 35 years old), the lower the ovarian reserve, and the less tolerant to chemotherapy, the risk of damage increases significantly.

[0005] With the increasing incidence of cancer in young patients (such as the increasing proportion of 30-40 year old patients in breast cancer and lymphoma patients) and the improvement of survival rate (the 5-year survival rate of some childhood cancers has exceeded 80%), fertility preservation and ovarian function protection have become an important part of clinical treatment, rather than an optional requirement. In 2006, the American Society of Clinical Oncology (ASCO) first published the Clinical Guidelines for Fertility Preservation in Cancer Patients, clearly stating that 'all patients of reproductive age should receive fertility preservation counseling before treatment', and recommending embryo cryopreservation as the preferred method. In 2018, ASCO updated the guidelines, including ovarian tissue cryopreservation as an 'acceptable clinical option', and emphasizing the need for earlier intervention for fertility preservation in pediatric patients. The European Society for Medical Oncology (ESMO) and the International Federation of Gynecology and Obstetrics (FIGO) have also published guidelines, both emphasizing the importance of individualized assessment—developing protection strategies based on tumor type, stage, patient age, and fertility needs.

[0006] In China, fertility preservation for female cancer patients started relatively late, but has developed rapidly in recent years. Institutions such as Peking Union Medical College Hospital and Fudan University Shanghai Cancer Center have carried out clinical applications of embryo freezing, ovarian tissue freezing, and other technologies; research on the protective mechanisms of GnRHa and the optimization of in vitro follicle culture technology has made some progress; the 2021 Chinese Expert Consensus on Fertility Preservation in Gynecological Malignancies was released, providing localized guidance for clinical practice. Despite this, the popularization of fertility preservation technology in China still faces challenges, such as insufficient patient awareness, high technology costs, and limited protection options for pediatric patients.

[0007] At present, the clinical exploration of ovarian function protection methods can be divided into "fertility preservation technology" and "ovarian function protection drugs" two categories, but its maturity and scope of application exist significant differences.

[0008] In the fertility preservation technology, embryo cryopreservation as the only mature technology, its process is through the ovulation to obtain oocytes, in vitro fertilization with sperm to form embryos and then cryopreservation, after the patient's recovery to thaw and transplant. The success rate of this technology is high (thawing and transplantation pregnancy rate is about 40%-50%), suitable for married and immediate fertility needs of patients. But the limitations are significant, need for spouse or sperm, not suitable for unmarried, no spouse or refuse to donate sperm women; ovulation needs 10-14 days, may delay the opportunity for chemotherapy, there is a risk for rapidly progressive tumors (such as some lymphoma) patients; estrogen rise caused by ovulation may stimulate hormone-sensitive tumors (such as ER-positive breast cancer) progression, limited clinical application.

[0009] Oocyte cryopreservation includes mature oocyte cryopreservation (need ovulation) and immature oocyte cryopreservation (no need for ovulation), suitable for unmarried women. But immature oocyte in vitro maturation technology is not mature, the pregnancy rate is only about 10%-20%, and it is currently used as an experimental technology.

[0010] Ovarian tissue cryopreservation is to remove part of the ovarian tissue before chemotherapy and transplant back to the body (in situ or ectopic) after treatment. This technology does not need ovulation, suitable for children, unmarried women and patients who need emergency chemotherapy, and is the main choice for children patients. But there are two major bottlenecks, the recovery rate of ovarian function after transplantation is low, about 50% of patients can restore menstruation, but the duration is short (average 2-5 years); the risk of tumor cell reimplantation, especially for patients with ovarian malignant tumors, the frozen tissue may contain cancer cells, leading to recurrence. Other technologies such as ovarian transposition surgery through surgery to move the ovaries out of the pelvic radiotherapy field, only suitable for patients with pelvic radiotherapy, ineffective for chemotherapy damage; ovarian transplantation (allogeneic) faces the problem of immune rejection, and the clinical application is very few.

[0011] In the field of medicine, gonadotropin-releasing hormone agonists (GnRHa) are the only clinically proven effective ovary protection drugs. Its mechanism of action is based on the regulation of the hypothalamic-pituitary-ovarian axis, the core mechanism is that GnRHa binds to pituitary GnRH receptors, initially stimulates the release of gonadotropins (FSH, LH) for a short period of time ('ignition effect'), and after continuous use, it leads to receptor desensitization, inhibits FSH secretion, and makes the follicle in a static state, reducing the sensitivity to chemotherapy drugs; The auxiliary mechanism includes reducing ovarian blood flow, reducing the exposure of chemotherapy drugs; Up-regulation of anti-apoptotic molecules (such as Bcl-2), inhibition of oocyte apoptosis; Maintain the level of anti-Müllerian hormone (AMH) to protect the follicle reserve. A number of randomized controlled trials have confirmed that GnRHa can reduce the incidence of POI, among breast cancer patients, the use of GnRHa can increase the rate of menstrual recovery by 30%-40% compared with the control group, and the success rate of fertility is increased by about 20%; Similar effects have been observed in patients with lymphoma and hematopoietic stem cell transplantation. More importantly, studies have found that GnRHa has no negative impact on tumor prognosis, even in hormone-sensitive tumors, it does not increase the risk of recurrence.

[0012] Although the existing technology provides options for ovary function protection, there are still many challenges in clinical practice, and breakthroughs in basic research may provide new ideas for solving these problems. The core difficulties of current clinical application include limited technical applicability, mature technology (such as embryo freezing) only covers a small number of patients, and the needs of children, unmarried, and emergency chemotherapy patients cannot be met; The safety of ovarian tissue freezing in tumor patients has not been completely solved, which limits its application in malignant tumor patients.

[0013] The timing of administration is critical for the optimal use of GnRHa, which should be initiated 1-2 weeks before chemotherapy to ensure follicular quiescence. However, patients with acute leukemia may not be able to wait, which may reduce the protective effect. In addition, the optimal dose, course of treatment, and compatibility with different chemotherapy regimens still need to be explored. Multidisciplinary cooperation is lacking, and the protection of ovarian function requires the cooperation of oncology, gynecology, and reproductive medicine. However, most hospitals have not yet established standardized processes, resulting in missed protection opportunities for patients. Future research directions include technological innovation, such as in vitro follicle culture technology, which can culture primitive follicles to mature oocytes in vitro, avoiding the risk of tumor transplantation. Stem cell therapy uses mesenchymal stem cells to repair ovarian damage, and animal experiments have shown that it can restore follicle reserves. Targeted drug delivery develops chemotherapy drug carriers targeting the ovary to reduce direct damage to follicles. Mechanism deepening of GnRHa, exploring the combination of GnRHa with other drugs (such as antioxidants and apoptosis inhibitors), whether it can enhance the protective effect; optimizing the dosing regimen for different types of tumors to improve individualized treatment. Accumulation of long-term follow-up data, current research focuses on short-term ovarian function recovery (such as menstrual recovery), but long-term safety (such as offspring health and tumor recurrence risk) data are still insufficient, and larger-scale long-term follow-up studies are needed.

[0014] Chemotherapy-induced ovarian damage is an important problem affecting the quality of life of female cancer patients. Its irreversibility and the trend of younger onset highlight the urgency of protection strategies. Currently, embryo cryopreservation technology provides some patients with the possibility of reproduction, and GnRHa, as the only clinically available protective drug, has shown good efficacy and safety in various tumors, making it an important choice for ovarian function protection. However, the gap between technical limitations and clinical needs still needs to be bridged through multidisciplinary cooperation and technological innovation. In the future, with the maturation of technologies such as in vitro follicle culture and stem cell therapy, and the optimization of GnRHa application schemes, it is hoped to provide individualized protection strategies for patients of different types and ages, ultimately achieving the dual goals of 'curing cancer' and 'preserving quality of life'. For clinicians, attention should be paid to patients' reproductive needs at the beginning of cancer treatment, and the optimal scheme should be developed based on guidelines and patient wishes, so that cancer survivors not only live longer, but also live better.

[0015] Dihydromyricetin (DMY) is a polyphenol compound found in medicinal and edible plants such as Ampelopsis megalophylla, Hovenia dulcis, and grape leaves. Ampelopsis megalophylla has the highest content of DMY and has significant antibacterial activity. It shows inhibitory effect on five foodborne pathogenic bacteria, including Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis, and Gram-negative bacteria such as Escherichia coli, Salmonella paratyphi, and Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) analysis shows that the antibacterial activity of DMY on Gram-negative bacteria is better than that of some synthetic antibacterial agents, which is related to the broad-spectrum activity of the hydroxyl groups (such as 5, 7, 3', 4', 5' positions) in its molecular structure. Acidic conditions (pH <7) enhance the membrane binding ability of DMY by protonating the hydroxyl groups, while high temperature or Ca 2+ metal ions can weaken its antibacterial effect through chelation or oxidative degradation.

[0016] Dihydromyricetin, molecular formula: C 15 H 12 O8, molecular weight: 320.25, SMILES code: O=C1[C@]([C@](C(C([H])=C2O[H])=C([H])C(O[H])=C2O[H])([H])OC3=C([H])C(O[H])=C([H])C(O[H])=C13)([H])O[H], chemical structure as follows:

[0017] .

[0018] No other literature or patents have reported other physiological activities and uses of the above-mentioned dihydromyricetin (DMY) so far. SUMMARY

[0019] Based on previous research, the applicant further explored the composition of the complex natural product for improving ovarian function damage and insulin resistance and found that the polyphenol component dihydromyricetin in medicinal and edible plants such as Ampelopsis megalophylla (tea vine), Hovenia dulcis, and grape leaves has a more significant effect on improving ovarian function damage.

[0020] The application discloses the use of dihydromyricetin DMY in the preparation of drugs for preventing and treating ovarian function damage and degeneration caused by chemotherapy drugs, and the chemical structure of dihydromyricetin DMY is as follows:

[0021] .

[0022] The dihydromyricetin DMY plays a role by activating GnRH.

[0023] The chemotherapy drug is cyclophosphamide.

[0024] The medicine is administered by subcutaneous injection.

[0025] The medicine is an injection solution consisting of dihydromyricetin DMY and pharmaceutically acceptable excipients, and can also be prepared into a freeze-dried preparation by freeze-drying process and the like.

[0026] The pharmaceutically acceptable excipients include, but are not limited to, freeze-drying protectants, isotonicity adjustors, antioxidants, pH adjustors, solvents.

[0027] The injection solution formula is as follows:

[0028] Dihydromyricetin DMY: 0.1g-1.0g

[0029] Sodium chloride: 8.0g

[0030] Ascorbic acid: 1.0g

[0031] 0.1M hydrochloric acid or sodium hydroxide solution: appropriate amount

[0032] Water for injection: constant volume to 1000ml

[0033] pH: 6.5-6.8

[0034] Total: 1000

[0035] The preparation method of the injection solution is as follows:

[0036] a) Take 70% of the prescription amount of water for injection, and dissolve sodium chloride and ascorbic acid;

[0037] b) Add dihydromyricetin DMY and dissolve;

[0038] c) Adjust the pH to 6.5-6.8;

[0039] d) Constant volume to the full amount;

[0040] e) Filter sterilization through 0.45μm and 0.22μm filter membranes;

[0041] f) Fill into ampoules, seal with nitrogen, and obtain.

[0042] The medicine has a protective effect on the endocrine function, reserve function and reproductive function of the ovary during and after chemotherapy.

[0043] The beneficial effects of the present application are as follows:

[0044] The present application first discloses the GnRH agonistic effect of dihydromyricetin DMY, and verifies the affinity and GnRH agonistic effect of dihydromyricetin DMY on GnRH receptor through in vitro experiments. Through model animal experiments, the present application further proves that dihydromyricetin DMY has a protective effect on the damage of ovarian function caused by chemotherapy. Experiments prove that, when using cyclophosphamide (CTX) for chemotherapy, dihydromyricetin DMY can not only reduce the damage of CTX to ovarian function during chemotherapy, but also plays a protective role in the recovery of ovarian function after drug withdrawal. This protective effect is not only reflected in the endocrine function and reserve function of the ovary, but also enables the reserved ovarian reserve function to achieve pregnancy after the end of chemotherapy, indicating that dihydromyricetin DMY also plays a protective effect on the reproductive function of the ovary. DETAILED DESCRIPTION

[0045] The present application will be further described in detail by the following examples, which are an explanation of the present application, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0046] Example 1 In vitro GnRH receptor binding capacity of dihydromyricetin DMY

[0047] The dihydromyricetin DMY solution (20 μL) with a concentration range of 1 × 10 -11 to 1 × 10 -5 M (n = 3) was moved into a 96-well plate, and the solution was prepared by a binding buffer (50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, 0.2% bovine serum albumin (BSA), adjusted to pH 7.4 with 1 M NaOH). 20 μL of ¹² 5 I-triptorelin binding buffer. Then, 60 μL of the binding buffer containing 10 μg of cell membrane (cell membrane expressing human gonadotropin-releasing hormone receptor (GnRHR)) was added to each well, and incubated at room temperature for 90 minutes. Subsequently, the cell membrane was sucked onto a glass fiber filter pretreated with 33% polyethyleneimine for 30 minutes, and then washed with 200 μL of washing buffer (50 mM HEPES, 500 mM NaCl, 0.1% BSA, adjusted to pH 7.4 with 1 M NaOH) for 5 times, and then washed with 100 μL of washing buffer for 15 times. After that, the filter membrane was cut into small pieces containing the cell membrane of each well, and the radioactivity bound to the cell membrane was measured using a gamma counter. The results show that the half inhibitory concentration (IC 50The GnRH affinity of DMY was 0.13±0.01 nM, and the GnRH affinity of triptorelin was 0.15±0.02 nM. According to the experimental results, DMY showed similar GnRH affinity to the control drug triptorelin.

[0048] Example 2 Protective effect of dihydromyricetin DMY on ovarian function injury of rats caused by chemotherapy

[0049] 2.1 Experimental animals: clean grade Wistar inbred female rats (body weight 200±20 g) were selected. Full pellet feed was fed, free feeding, room temperature was controlled between 20℃ to 25℃, relative humidity was maintained at 45% to 55%, daily light time was 12 hours. After adaptive feeding for one week, vaginal secretions were collected every morning at 9:00, smears were made and observed under a light microscope to observe the sexual cycle, and animals with normal sexual cycle were selected for the experiment.

[0050] 2.2 Animal grouping: the qualified female rats were randomly divided into the following groups:

[0051] (1) Blank control group (10): no treatment.

[0052] (2) CTX group (12): the first loading dose was CTX 50 mg / (kg·d), intraperitoneal injection, lasting for 1 day; then CTX 8 mg / (kg·d) was intraperitoneally injected once a day, continuously for 14 days.

[0053] (3) GnRHa positive control group (12): triptorelin acetate was dissolved in 0.5 ml of 0.9% sodium chloride injection for subcutaneous injection, the loading dose was 52.5 μg / (kg·d), continuously for 7 days, then 10.5 μg / (kg·d) was used as the maintenance dose, continuously for 18 days, for a total of 25 days.

[0054] (4) CTX+GnRHa positive control group (12): the amount, method and time of triptorelin were the same as those of the GnRHa positive control group, and the first loading dose, the maintenance dose and the method were consistent with those of the CTX group and the GnRHa positive control group. CTX was used on the first day of triptorelin injection.

[0055] (5) CTX+low-dose dihydromyricetin DMY group (12): dihydromyricetin DMY was dissolved in 0.5 ml of 0.9% sodium chloride injection for subcutaneous injection, the loading dose was 5 μg / (kg·d), continuously for 7 days, then 1 μg / (kg·d) was used as the maintenance dose, continuously for 18 days, for a total of 25 days. CTX was used on the first day of administration, and the first loading dose, the maintenance dose and the method of CTX were the same as those of the CTX group.

[0056] (6) CTX + dihydroquercetin DMY middle dose group (12): the administration method of dihydroquercetin DMY is the same as that of the low dose group, and the loading dose and the maintenance dose are 10 μg / (kg·d) and 2 μg / (kg·d) respectively. On the first day of administration, CTX is added, and the first loading dose and the maintenance dose of CTX and the usage are the same as those of the CTX group.

[0057] (7) CTX + dihydroquercetin DMY high dose group (12): the administration method of dihydroquercetin DMY is the same as that of the low dose group, and the loading dose and the maintenance dose are 20 μg / (kg·d) and 4 μg / (kg·d) respectively. On the first day of administration, CTX is added, and the first loading dose and the maintenance dose of CTX and the usage are the same as those of the CTX group.

[0058] 2.3 Test observation and sampling:

[0059] FSH and E2:

[0060] Each group of animals is observed for the estrous cycle by vaginal smear every day during the medication period. The blank control group is measured for FSH and E2 (follicle-stimulating hormone and estradiol) in the serum of rats at each time point before medication, at the end of medication, 1 week and 2 weeks after medication by radioimmunoassay, and the rest of the groups are measured for FSH and E2 in the serum of rats every other estrous cycle during the medication period and after medication until 2 weeks after medication to observe the changes.

[0061] After the end of medication, 5 rats are sacrificed in each group during the estrous interval, and blood is taken by heart puncture. The bilateral ovaries are removed and weighed, and then fixed with 4% paraformaldehyde for subsequent use.

[0062] The remaining animals continue to be observed for the estrous cycle by vaginal smear, and 5 rats in each group are sacrificed again after two weeks, and blood is taken, weighed and fixed in the same way. After paraffin embedding, the bilateral ovaries are cut into 5 μm sections and stained with HE, and 1 out of every 10 sections is observed under a light microscope to observe the pathological changes of the ovarian tissue sections and count the number of follicles with normal follicle structure (the number of follicles in the bilateral ovaries is added).

[0063] The remaining 2 rats in each of the GnRHa positive control group, the CTX group, the CTX + GnRHa positive control group, the CTX + dihydroquercetin DMY low dose group, the CTX + dihydroquercetin DMY middle dose group, and the CTX + dihydroquercetin DMY high dose group are mated with Wistar inbred male rats to observe the pregnancy.

[0064] 2.4 Radioimmunoassay of rat serum follicle-stimulating hormone and estradiol:

[0065] According to the instructions, I-FSH and 125 I-FSH and 125I-E2 radioimmunoassay kit for determination.

[0066] 2.5 Pathological section making:

[0067] (1) After the rats were killed, the abdomen was dissected, and the bilateral ovaries were found along the bilateral oviducts.

[0068] (2) The bilateral ovaries were removed, weighed, and placed in 4% paraformaldehyde for fixation and stored in a 4°C refrigerator.

[0069] (3) After the ovaries were embedded in paraffin, 5 μm sections were cut and stained with HE, and one section out of every 10 sections was observed under a light microscope.

[0070] (4) The pathological changes of the ovarian tissue sections were observed and compared under a light microscope, and the number of follicles with normal follicle structure was counted (the number of follicles in both ovaries was added). The number of primordial follicles, growing follicles, and mature follicles was counted. The follicle classification standard was: primordial follicle < 30 μm, growing follicle 30 μm - 45 μm, and mature follicle > 45 μm.

[0071] 2.6 Experimental results

[0072] 2.6.1 Changes in the estrous cycle of rats

[0073] According to the standard in Table 1, the changes in the estrous cycle of animals in each group were evaluated.

[0074] Table 1 Cytological changes in the estrous cycle of female Wistar rats

[0075] .

[0076] Table 2 Description of the estrous cycle of experimental animals in each group

[0077]

[0078] 2.6.2 Changes in the serum FSH and E2 concentrations of rats:

[0079] Table 3 Changes in the FSH and E2 concentrations of experimental animals in each group

[0080] .

[0081] Based on the data in the above table, the following conclusions can be drawn:

[0082] 1. The FSH and E2 concentrations in the serum of rats in the blank control group did not change significantly before treatment, 15 days after treatment, at the time of drug withdrawal, 1 week after drug withdrawal, and 2 weeks after drug withdrawal.

[0083] 2. The FSH of CTX group increased and the E2 decreased after 15 days of administration. There was a significant difference compared with the blank control group at the time of drug withdrawal. After drug withdrawal, the FSH concentration in the serum of the CTX group continued to rise, and the serum E2 concentration continued to decrease, which was significantly different compared with the blank control group at the same period.

[0084] 3. The FSH and E2 concentrations in the serum of the GnRHa group slightly increased after 15 days of administration, then decreased, and reached the lowest point at the time of drug withdrawal, which was significantly different compared with the blank control group. After drug withdrawal, the FSH and E2 concentrations in the serum began to rise, and after 1 week of drug withdrawal, the FSH and E2 concentrations in the serum of the GnRHa group returned to normal levels, which was not significantly different compared with the blank control group.

[0085] 4. The FSH concentration in the serum of the CTX+GnRHa positive control group changed little from before administration to 2 weeks after administration. The E2 concentration continued to decrease, and there was a significant difference compared with the blank control group at the time of drug withdrawal. At 2 weeks after drug withdrawal, the serum E2 concentration increased close to the normal level, and there was no significant difference compared with the blank control group.

[0086] 5. The FSH of the CTX+Dihydromyricetin DMY low, medium and high dose groups slightly increased after 15 days of administration, and the increase decreased with the increase of the dose of Dihydromyricetin DMY. It is worth noting that the increase of the CTX+Dihydromyricetin DMY medium dose group was similar to that of the CTX+GnRHa positive control group, and the FSH returned to normal level after drug withdrawal and in the subsequent 1 week and 2 weeks, which was not significantly different compared with before administration. The E2 level significantly decreased after 15 days of administration, and the decrease decreased with the increase of the dose of Dihydromyricetin DMY. The E2 level continued to decrease at the time of drug withdrawal, and the decrease of the CTX+Dihydromyricetin DMY medium dose group was similar to that of the CTX+GnRHa positive control group. The E2 level rose after 1 week of drug withdrawal, and the rise increased with the increase of the dose of Dihydromyricetin DMY after 2 weeks of drug withdrawal. The E2 level of the CTX+Dihydromyricetin DMY high dose group rose to the level before administration, and the E2 levels of the low and medium dose groups were slightly lower than before administration. The rise of the CTX+Dihydromyricetin DMY medium dose group was similar to that of the CTX+GnRHa positive control group.

[0087] 2.6.3 Changes in the weight of rat ovaries and the number of follicles:

[0088] Table 4 Changes in the number of follicles and the weight of ovaries in rats of each group (N=5)

[0089] .

[0090] 1. The bilateral ovary weight of the blank control group had no significant change at the time of drug withdrawal and two weeks later. After administration, the bilateral ovary weight of the CTX group of rats continued to decrease, and two weeks after drug withdrawal, it continued to decrease, which was extremely significantly different from the blank control group. The bilateral ovary weight of the GnRHa group, the CTX+GnRHa positive control group, and the CTX+Dihydromyricetin DMY low, medium and high dose groups of rats were significantly reduced at the time of drug withdrawal, and then gradually recovered. Two weeks after drug withdrawal, the GnRHa group had no difference with the blank control group; the recovery degree of the CTX+GnRHa positive control group, the CTX+Dihydromyricetin DMY low, medium and high dose groups increased with the increase of the dose of Dihydromyricetin DMY, among which the CTX+Dihydromyricetin DMY medium dose group was equivalent to the CTX+GnRHa positive control group, and the CTX+Dihydromyricetin DMY high dose group was equivalent to the blank control group.

[0091] 2. The total number of follicles and the number of primordial follicles of the blank control group had no obvious change at the time of drug withdrawal and two weeks later. After administration, the total number of follicles of the CTX group of rats continued to decrease, which was significantly less than the blank control group at two weeks after drug withdrawal, and the difference between the two groups was significant.

[0092] The total number of follicles of the GnRHa positive control group was slightly more than that of the blank control group at two weeks after drug withdrawal. At the time of drug withdrawal and two weeks after drug withdrawal, the number of primordial follicles of the GnRHa positive control group was still more than that of the blank control group, and showed a gradual downward trend. At two weeks after drug withdrawal, the total number of follicles of the CTX+GnRHa positive control group was less than that of the blank control group, but was significantly more than that of the CTX group, and the difference between the CTX group and the CTX+GnRHa positive control group was significant. At the time of drug withdrawal, the number of primordial follicles was slightly more than that of the blank control group; at two weeks after drug withdrawal, the number of primordial follicles was close to that of the blank control group.

[0093] The total number of follicles and the number of primordial follicles of the CTX+Dihydromyricetin DMY low, medium and high dose groups were close to those of the blank control group at the time of drug withdrawal, and increased with the increase of the dose of Dihydromyricetin DMY. Two weeks after drug withdrawal, the number of primordial follicles and the total number of follicles decreased more obviously than at the time of drug withdrawal, but the decrease decreased with the increase of the dose of Dihydromyricetin DMY. The total number of follicles of the CTX+Dihydromyricetin DMY high dose group even increased slightly compared with that at the time of drug withdrawal. The number of primordial follicles and the total number of follicles of the CTX+Dihydromyricetin DMY medium dose group were equivalent to those of the CTX+GnRHa positive control group. This proves that Dihydromyricetin DMY has significant GnRH agonist activity, and at the medium dose level, it is equivalent to the GnRH positive control group (triptorelin).

[0094] 2.6.4 Pregnancy of rats

[0095] GnRHa group, CTX+GnRHa positive control group, CTX+Dihydromyricetin DMY low, medium and high dose group, respectively, 1 female rat successfully pregnant, the gestation time was 30 days after drug withdrawal (equivalent to 6-7 normal estrous cycle of rats), 49 days after drug withdrawal (equivalent to 10-12 normal estrous cycle of rats), 56 days after drug withdrawal, 46 days after drug withdrawal, 42 days after drug withdrawal, CTX group of rats were not pregnant. This proves that dihydromyricetin DMY has a certain improvement and repair effect on the ovarian damage caused by CTX chemotherapy, similar to the positive control drug triptorelin.

[0096] 2.7 Summary

[0097] 1. CTX mainly damages growing follicles and mature follicles.

[0098] 2. GnRHa has an inhibitory effect on FSH and E2 after continuous administration, and FSH and E2 can return to normal after drug withdrawal.

[0099] 3. GnRHa has the effect of recruiting primordial follicles.

[0100] 4. The use of GnRHa during CTX administration chemotherapy not only reduces the damage of CTX to ovarian function during chemotherapy, but also has a protective effect on subsequent ovarian damage after drug withdrawal. The protective effect is reflected in the endocrine function and reserve function of the ovary, and the preserved ovarian reserve function can also result in pregnancy after chemotherapy, indicating that the reproductive function of the ovary is also protected.

[0101] 5. Dihydromyricetin DMY at low, medium and high dose levels, as a GnRH agonist, combined with CTX, can improve the damage caused by CTX chemotherapy, and the protective effect of the dose level is equivalent to that of the positive drug triptorelin.

[0102] Example 3 Preparation of dihydromyricetin DMY injection

[0103] Table 5 Dihydromyricetin DMY injection formula

[0104]

[0105] Preparation method:

[0106] Step 1) Take 70% of the prescription amount of water for injection, add the prescription amount of sodium chloride and ascorbic acid, and stir to dissolve;

[0107] Step 2) Take the prescription amount of dihydromyricetin DMY, add the solution obtained in step 1), and stir to dissolve;

[0108] Step 3) Take the liquid obtained in step 2), adjust the pH to 6.5-6.8 with 0.1M hydrochloric acid or sodium hydroxide solution;

[0109] Step 4) The resulting solution in step 3) was diluted with water for injection to the full volume;

[0110] Step 5) The resulting solution in step 4) was filtered through 0.45 μm and 0.22 μm microporous filter membranes in sequence to remove bacteria;

[0111] Step 6) The resulting solution in step 5) was filled into a glass ampoule with appropriate volume, sealed after nitrogen filling, and obtained.

[0112] The resulting injection solution is a concentrated solution, which can be further diluted with a suitable solvent as needed.

[0113] The above invention content and examples illustrate the core principles, main characteristics and advantages of the present patent application. It should be understood by those skilled in the art that the present patent application is not limited by the above examples, and the above examples and the mentioned in the specification are only the best technical solutions of the present patent application. There are many changes and optimizations of the present patent application without deviating from the purpose and scope of the present patent application. That is, the use of dihydromyricetin DMY and its pharmaceutical composition for preparing drugs for treating or preventing chemotherapy-induced ovarian function damage, all belong to the scope of protection of the present patent application, and the scope of protection of the present patent application is determined by the appended claims and their equivalents.

Claims

1. The use of dihydromyricetin (DMY) in the preparation of drugs for the prevention and treatment of ovarian dysfunction and degeneration caused by chemotherapy drugs, characterized in that, The dihydromyricetin DMY has the following chemical structure: 。 2. The use as described in claim 1, characterized in that, The chemotherapy drug is cyclophosphamide.

3. The use as described in claim 1, characterized in that, The dihydromyricetin DMY exerts its protective effect by activating GnRH receptors.

4. The use as described in claim 1, characterized in that, The drug is administered by injection.

5. The use as described in claim 4, characterized in that, The injection method is subcutaneous injection.

6. The use as described in claim 5, characterized in that, The drug is composed of dihydromyricetin (DMY) and pharmaceutically acceptable excipients, and is prepared into a lyophilized formulation by a freeze-drying process.

7. The use as described in claim 6, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: lyophilization protectants, isotonic modifiers, antioxidants, pH adjusters, or solvents.

8. The use as described in claim 4, characterized in that, The drug is an injectable solution, and its formulation includes: Dihydromyricetin (DMY): 0.1g-1.0g Sodium chloride: 8.0g Ascorbic acid: 1.0g 0.1M hydrochloric acid or sodium hydroxide solution: Adjust the pH to 6.5-6.

8. Water for injection: bring volume to 1000ml A total of 1000 vials of injection solution were produced.

9. The use as described in claim 8, characterized in that, The method for preparing the injection solution includes the following steps: a) Dissolve sodium chloride and ascorbic acid in 70% of the prescribed amount of water for injection; b) Add dihydromyricetin DMY and dissolve it; c) Adjust the pH to 6.5-6.8; d) Bring to full volume; e) Sterilization is achieved through filtration using 0.45μm and 0.22μm filter membranes; f) Fill the ampoules with nitrogen, seal them, and you have the product.

10. The use as described in claim 1, characterized in that, The drug has a protective effect on ovarian endocrine function, reserve function, and fertility during and after chemotherapy.

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