Active molecules and derivatives thereof for use in ovarian dysfunction diseases

Abstract

The present disclosure relates to active molecules and derivatives thereof for use in ovariogenic diseases. In particular, the present disclosure relates to behenic acid or a derivative thereof or a pharmaceutically acceptable salt thereof, a compound of Formula I or a pharmaceutically acceptable salt thereof, or the use of behenic acid or a derivative thereof or a pharmaceutically acceptable salt thereof in combination with a compound of Formula I or a pharmaceutically acceptable salt thereof in the treatment or prevention of ovariogenic diseases.

Description

Technical Field

[0001] This invention relates to the field of medicine, and in particular to active molecules and their derivatives for the prevention or treatment of ovarian dysfunction. Background Technology

[0002] The ovary is an important reproductive and endocrine organ in female animals, whose functions include ovulation and hormone secretion (such as steroid hormones, hormones, and progesterone). When ovarian function is disordered, such as abnormal follicle maturation or decreased steroid hormone secretion, it can lead to a series of ovarian dysfunction diseases, such as follicle development, maturation, or ovulation disorders. Patients may experience infertility, miscarriage, and premature ovulation, and the endocrine function of the ovary may also be affected.

[0003] Currently, hormone replacement therapy (HRT) is the primary treatment for ovarian dysfunction in clinical practice. HRT offers rapid short-term relief and effectively improves estrogen and follicle-stimulating hormone (FSH) levels. However, it has numerous side effects and contraindications, such as gastrointestinal discomfort, weight gain, and increased risks of venous thrombosis, endometrial cancer, and breast cancer. Clinically, patients often experience psychological burden associated with hormone therapy or refuse it altogether. Patients desiring fertility often resort to assisted reproductive technologies (ART), which are expensive and often ineffective for patients with anovulation or infrequent ovulation. Importantly, there are still no clearly defined and effective strategies for preventing or delaying the progression of ovarian dysfunction.

[0004] Therefore, there is an urgent need to find a new drug for the prevention or treatment of ovarian dysfunction, which has advantages such as significant efficacy and low side effects. Summary of the Invention

[0005] To address one of the aforementioned technical problems in the prior art, this disclosure provides a drug and a treatment method for treating or preventing ovarian dysfunction. Drug treatment according to this disclosure can effectively restore ovarian function and promote follicle development and ovulation.

[0006] According to one aspect of this disclosure, the use of behenic acid, or a derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment or prevention of ovarian dysfunction is provided.

[0007] According to some implementation methods, the derivatives of behenic acid can be behenic acid glycerides, such as behenic acid monoglycerides, behenic acid diglycerides, or behenic acid triglycerides.

[0008] According to another aspect of this disclosure, the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of ovarian dysfunction is provided.

[0009] Where n is an integer selected from 2 to 4. For example, n can be 2, 3 or 4.

[0010] According to some embodiments, the compound represented by Formula I is selected from 2-amino-4-(carbamoylamino)butyric acid, 2-amino-5-(carbamoylamino)valerate (citrulline), or 2-amino-6-(carbamoylamino)hexanoic acid (homocitrulline). According to some embodiments, the compound represented by Formula I is selected from citrulline or homocitrulline. According to some embodiments, the compound represented by Formula I is selected from L-homocitrulline.

[0011] According to another aspect of this disclosure, the use of behenic acid or a derivative thereof or a pharmaceutically acceptable salt thereof, and a combination of the compound represented by Formula I above or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment or prevention of ovarian dysfunction is provided.

[0012] According to some implementation methods, the ovarian dysfunction may include, but is not limited to, premature ovarian insufficiency (POI), premature ovarian failure (POF), diminished ovarian reserve (DOR), poor ovarian response (POR), early menopause, ovarian dysfunction, insufficient ovarian function, and low ovarian function. The ovarian dysfunction may be caused by genetic factors, iatrogenic factors, immunological factors, environmental factors, advanced physiological age, or other factors. The ovarian dysfunction may present with symptoms such as hormonal imbalances, menstrual cycle irregularities, infrequent ovulation, or anovulation, and can lead to decreased reproductive function, infertility, perimenopausal syndrome, menopausal syndrome, and other diseases.

[0013] According to a specific implementation, the ovarian dysfunction is premature ovarian insufficiency (POI). According to a specific implementation, the ovarian dysfunction is diminished ovarian reserve (DOR). According to a specific implementation, the ovarian dysfunction is premature ovarian failure (POF). According to a specific implementation, the ovarian dysfunction is poor ovarian response (POR). According to a specific implementation, the ovarian dysfunction is early menopause. According to a specific implementation, the ovarian dysfunction is ovarian dysfunction. According to a specific implementation, the ovarian dysfunction is insufficient ovarian function. According to a specific implementation, the ovarian dysfunction is hypofunction.

[0014] According to specific embodiments, behenic acid or its derivatives or pharmaceutically acceptable salts can prolong the estrous cycle in patients with ovarian insufficiency. According to specific embodiments, behenic acid or its derivatives or pharmaceutically acceptable salts can restore ovulation, for example, by increasing the number of ovulations in patients with ovarian insufficiency. According to specific embodiments, behenic acid or its derivatives or pharmaceutically acceptable salts can reduce oxidative stress levels in patients with ovarian insufficiency.

[0015] According to specific embodiments, the compound represented by Formula I above, or a pharmaceutically acceptable salt thereof, can prolong the estrous cycle in patients with ovarian insufficiency. According to specific embodiments, the compound represented by Formula I above, or a pharmaceutically acceptable salt thereof, can restore ovulation, for example, by increasing the number of ovulations in patients with ovarian insufficiency. According to specific embodiments, the compound represented by Formula I above, or a pharmaceutically acceptable salt thereof, can reduce oxidative stress levels in patients with ovarian insufficiency.

[0016] According to specific embodiments, behenic acid or its derivatives or pharmaceutically acceptable salts, used in combination with the compound shown in Formula I or its pharmaceutically acceptable salts, can prolong the estrous cycle in patients with ovarian insufficiency. According to specific embodiments, behenic acid or its derivatives or its pharmaceutically acceptable salts, used in combination with the compound shown in Formula I or its pharmaceutically acceptable salts, can restore ovulation, for example, increasing the number of ovulations in patients with ovarian insufficiency. According to specific embodiments, behenic acid or its derivatives or its pharmaceutically acceptable salts, used in combination with the compound shown in Formula I or its pharmaceutically acceptable salts, can reduce the level of oxidative stress in patients with ovarian insufficiency.

[0017] According to another aspect, a composition is provided comprising behenicol or a derivative thereof or a pharmaceutically acceptable salt thereof, and / or a compound represented by Formula I above or a pharmaceutically acceptable salt thereof.

[0018] According to some embodiments, the composition further comprises a pharmaceutically acceptable excipient. According to some embodiments, the pharmaceutically acceptable excipient may include, but is not limited to, one or more of a pharmaceutical carrier, diluent, adjuvant, and excipient.

[0019] According to some embodiments, the dosage form of the composition includes, but is not limited to, tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release formulations, controlled-release formulations, or targeted formulations.

[0020] According to some embodiments, the composition can be administered, for example, by injection, oral administration, rectal administration, etc.

[0021] According to some embodiments, the behenic acid or its derivatives or pharmaceutically acceptable salts, and / or the compound represented by Formula I above or its pharmaceutically acceptable salts, are formulated for administration in the same dosage form or in separate dosage forms.

[0022] According to another aspect, a method for treating or preventing ovarian disorders is provided, the method comprising administering to a subject in need a therapeutically effective amount of behenic acid or a derivative thereof or a pharmaceutically acceptable salt thereof, and / or a compound represented by Formula I above or a pharmaceutically acceptable salt thereof.

[0023] According to some embodiments, the method includes simultaneously or sequentially applying behenic acid or a derivative thereof or a pharmaceutically acceptable salt thereof, and / or the compound represented by Formula I above or a pharmaceutically acceptable salt thereof.

[0024] The inventors of this disclosure compared fasting serum from patients with ovarian insufficiency with those from healthy individuals (controls) and found that the levels of metabolites such as behenic acid and homocitrulline were significantly reduced in the serum of patients with ovarian insufficiency. This disclosure further utilized a mouse model of premature ovarian failure to verify that oral administration of behenic acid, homocitrulline, or a combination of behenic acid and homocitrulline, via gavage, resulted in weight recovery, prolonged estrous cycles, increased ovulation count, and reduced oxidative stress in mice within just two weeks. These results indicate that behenic acid, homocitrulline, or a combination of behenic acid and homocitrulline have a significant therapeutic effect on ovarian disorders.

[0025] In existing technologies, hormonal drugs are commonly used to treat ovarian disorders. However, hormones have several side effects. First, hormones can stimulate endometrial lesions. A woman's estrogen, androgen, and progesterone interact and regulate each other. If estrogen is secreted excessively under the influence of drugs and the pituitary gland, the endometrium will experience abnormal proliferation due to long-term hormone exposure, leading to endometrial lesions. Second, long-term exposure to hormones can lead to uterine fibroids, and breast diseases, such as breast hyperplasia and even breast cancer, can also occur. Furthermore, estrogen can affect the central nervous system; excessive estrogen intake can cause dizziness, nausea, and even hypertension and diabetes. In contrast, the behenicol disclosed in this invention is a docosanoic acid, a long-chain saturated fatty acid naturally found in peanuts, most seed oils, animal milk fat, and marine oils. The compounds represented by Formula I (such as citrulline or homocitrulline) are themselves metabolites in humans and mammals. Therefore, using these two naturally occurring substances to treat ovarian disorders will not produce adverse reactions, is safe, and has few side effects. Attached Figure Description

[0026] Figure 1 A schematic diagram of a mouse drug administration and experimental design process according to one embodiment is shown.

[0027] Figure 2 The changes in body weight of mice before, after and after modeling, according to one embodiment are shown.

[0028] Figure 3 shows the results of estrous cycle determination in mice according to one implementation method. Figure 3A This is a diagram of the estrous cycle in mice; Figure 3B Stacked plot of the proportions of mice in each group at different stages of the estrous cycle.

[0029] Figure 4 The effect of drug administration on the number of eggs retrieved in mice according to one embodiment is shown. Figure 4 A is a representative diagram of oocytes expelled from the control group and the combined treatment group, respectively; Figure 4 B is a graph showing the average number of ovulations in each group.

[0030] Figure 5 The results of oxidative stress level determination in mice after administration according to one embodiment are shown. Figure 5 A shows the serum malondialdehyde (MDA) level in mice after drug administration; Figure 5 B shows the serum superoxide dismutase (SOD) level in mice after drug administration; Figure 5 C shows the serum glutathione (GSH) level in mice after administration.

[0031] Figure 6 shows a slice of an ovary from a mouse according to one embodiment and the results of follicle classification and counting. Figure 6A A slice of a mouse ovary is shown; Figure 6B The results of mouse follicle classification and counting are shown.

[0032] Figure 7 The diagram shows the differentially expressed genes in three treatment groups compared to the control group according to one implementation method, as well as the intersection of the three treatment groups. Figure 7 A shows the number and percentage of upregulated genes in the three treatment groups; Figure 7 B shows the number and percentage of downregulated genes in the three treatment groups.

[0033] Figure 8 Functional enrichment maps of differentially expressed genes that were commonly upregulated or downregulated in three treatment groups according to one implementation method are shown. Figure 8 A shows the functional enrichment map of genes that were commonly upregulated in the three treatment groups; Figure 8 B shows a functional enrichment map of genes that were downregulated in all three treatment groups.

[0034] Figure 9 The diagram shows signaling pathway enrichment of differentially regulated genes that were commonly upregulated or downregulated in three treatment groups according to one implementation method. Figure 9A shows a KEGG plot of the gene that was upregulated in all three treatment groups; Figure 9 B shows a KEGG plot of the genes that were downregulated in all three treatment groups.

[0035] Figure 10 A heatmap of the upregulated or downregulated top genes in three treatment groups according to one implementation method is shown. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0037] definition

[0038] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0039] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0040] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0041] The term "premature ovarian insufficiency" used in this article refers to a serious ovarian dysfunction that refers to the loss of ovarian function in women before the age of 40 due to follicle depletion. Clinical manifestations include amenorrhea for four consecutive months, oligomenorrhea or polymenorrhea, with two follicle-stimulating hormone (FSH) levels exceeding 25 U / L at intervals of more than four weeks, with or without fluctuating estrogen levels. Long-term risks include congenital heart disease, intellectual disability, mood disorders, osteoporosis, adrenal and thyroid hypofunction, diabetes, and recurrent miscarriage. POI patients experience severe ovulation disorders due to ovarian dysfunction, resulting in a significant decrease or loss of fertility. The incidence of POI in patients with primary amenorrhea is as high as 10-28%, with an overall incidence of approximately 3.7%, and it shows a trend of increasing annually. Clinically, POI patients may present with primary amenorrhea or even absence of puberty. Most cases are secondary amenorrhea, caused by natural aging, chromosomal or gene defects, autoimmune diseases, environmental factors, and iatrogenic factors.

[0042] The term "premature ovarian failure" used in this article refers to a woman under the age of 40 experiencing amenorrhea for more than 4 to 6 months, with two follicle-stimulating hormone (FSH) levels exceeding 40 U / L at intervals of more than 4 weeks, accompanied by decreased estrogen and menopausal symptoms. Premature ovarian failure is one of the leading causes of female infertility. The American Society for Reproductive Medicine uses FSH levels, fertility, and menstrual status as parameters to classify the disease progression into stages such as normal, occult, biochemically abnormal, and clinically abnormal.

[0043] The term "ovarian reserve" as used in this article refers to the number of primordial follicles contained in the ovarian cortex of a woman, which does not increase after birth, and the number of primordial germ cells in the ovarian cortex does not increase further. Diminished / decreased ovarian reserve (DOR), also known as low ovarian reserve, is when the number of follicles in the ovaries is less than the expected number, resulting in decreased ovarian responsiveness and fertility in women of reproductive age. This makes conception more difficult and reduces the chances of receiving in vitro fertilization (IVF) and other fertility treatments. DOR also leads to a higher miscarriage rate compared to women without it. Women with DOR have a decreased number and / or quality of oocytes in their ovaries, accompanied by elevated FSH levels.

[0044] The term “early menopause” as used in this article refers to menopause occurring before the normal age of menopause, specifically before the age of 45.

[0045] The term "poor ovarian response" used in this article, also known as poor ovarian function, refers to a woman's ovaries responding poorly to stimulation drugs and retrieving fewer eggs during controlled ovarian stimulation using assisted reproductive technology. Poor ovarian response is considered an early sign of declining ovarian reserve.

[0046] The term "estrogen" used in this article primarily refers to hormones secreted by the ovaries, including estradiol (E2), estrone, and estriol. Estrogens promote the development and maturation of accessory reproductive organs, the vagina, uterus, and fallopian tubes in adolescent girls. Estradiol itself is a steroidal estrogen secreted by the granulosa cells of the follicles in the ovary. Estradiol exists in two forms, α and β, with α-type E2 having a stronger physiological effect. In patients with premature ovarian failure, E2 levels are decreased.

[0047] The term "iatrogenic factors" used in this article refers primarily to ovarian dysfunction caused by surgery, radiotherapy, and chemotherapy drugs, such as ovarian damage.

[0048] As used herein, the term "pharmaceutically acceptable" means that it can be administered to humans and / or other animals as subjects without producing excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to auxiliary materials that coexist with the active ingredient in a pharmaceutical preparation without producing excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH adjusters, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceuticalally acceptable excipient" refers to a highly safe excipient suitable for a specific pharmaceutical preparation and routinely used in pharmaceutical practice. The term "carrier" includes, but is not limited to, liposomes, liposomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0049] As used herein, the term “pharmaceutically acceptable salt” may include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., quaternary ammonium salts).

[0050] The term "estrous cycle," as used in this article, refers to a recurring physiological cycle in female placental mammals, induced by sex hormones. In mice and rats, the estrous cycle repeats rhythmically every 4–5 days. Based on the follicular development pattern and reproductive endocrine changes during the estrous cycle, it can be divided into proestrus, estrus, metestrus, and diaestrus. Mice and rats are widely used as animal models to study the estrous cycle in experiments exploring the mechanisms of diseases related to the female menstrual cycle, evaluating drug efficacy, and developing corresponding drugs.

[0051] The term "prevention" as used in this article refers to preventive treatment of subclinical disease states, aimed at reducing the probability of clinical disease states occurring. "Prevention" can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of subjects who have not yet presented with a clinical disease state, while secondary prevention is defined as prevention of the recurrence of the same or similar clinical disease state.

[0052] As used in this article, the term "treatment" refers to the treatment of a disease, symptom, or condition, including: 1) suppressing the development of a disease, symptom, or condition; and / or, 2) delaying or alleviating a disease, symptom, or condition.

[0053] Biomarkers mentioned herein, such as anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), can be detected using methods generally known in the art. Detection methods typically encompass those that quantify the levels of biomarkers in a sample (quantitative methods). Which of the following methods are suitable for the qualitative and / or quantitative detection of biomarkers is generally known to those skilled in the art. Samples can be readily analyzed, for example, using immunoassays such as ELISA, RIA, etc., for proteins, and are commercially available.

[0054] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0055] Example

[0056] Example 1. Method for preparing a POI model and treatment using behenicol and / or L-hocitrulline.

[0057] 1. Preparation of animal models: Thirty-four 8-week-old female C57 mice were selected and injected intraperitoneally once a day with cyclophosphamide (SIGMA) at a dose of 120 mg / kg body weight and busulfan (Aladdin) at a dose of 30 mg / kg body weight for one week to establish an ovarian insufficiency (POI) model.

[0058] 2. Grouping: Twenty-five POI model mice were randomly divided into four groups: behenic acid (Doc) treatment group (n=9), L-homocycutaine (Hcit) treatment group (n=6), behenic acid and holocycutaine (Doc+Hcit) treatment group (n=6), and saline group (n=7) as control group. Figure 1A schematic diagram of the experimental procedure is shown. Simultaneously with the establishment of the POI model, mice in each group were treated with medication. The L-homocytrulline treatment group, the behenicol treatment group, and the L-homocytrulline + behenicol combination group were administered 10 mg / kg / d L-homocytrulline, 100 mg / kg / d behenicol, 100 mg / kg / d behenicol, and 10 mg / kg / d L-homocytrulline mixture, respectively, via gavage at a dose of 0.1 mL / 10 g. The control group mice were administered an equal volume of physiological saline orally via gavage daily. Treatment continued for 14 days. Daily behavioral observations and body weight measurements were performed on the mice, and vaginal cytology smears were prepared daily to observe changes in the estrous cycle.

[0059] 3. Perform vaginal smear cytology observation daily.

[0060] Expose the mouse vagina, instill approximately 30 μL of physiological saline, gently aspirate it onto a glass slide, and observe under a microscope. Staining may be necessary: ​​fix with 95% ethanol for 10 minutes, stain with hematoxylin aqueous solution for 5–8 minutes, rinse with running water to separate the colors, then stain with eosin for 3 minutes. After drying, mount with neutral resin.

[0061] 4. Weigh the patient before modeling (at the start of treatment), after modeling, and after treatment.

[0062] 5. After 14 days of drug administration, some mice in each group were sacrificed, and their blood was collected. After standing, the blood was centrifuged at 4500 rpm to obtain serum, which was stored at -80℃ for later use to determine hormones such as follicle-stimulating hormone and oxidative stress indicators such as superoxide dismutase (SOD) in mice.

[0063] Example 2. Results of weight measurement

[0064] The body weight of POI model mice was measured before modeling, after modeling, and after treatment in each group.

[0065] The weight measurements of the 34 mice in Example 1 are shown in Table 1 below.

[0066] Table 1. Body weight of mice in each group in Example 1 before modeling (week 0), after modeling (week 1), and after treatment (week 2).

[0067]

[0068] Repeated measures ANOVA was performed three times: before modeling, after modeling, and after treatment.

[0069] Within-group comparison: The test of sphericity showed P = 0.157 (>0.05), satisfying the sphericity assumption. Therefore, the results of the univariate ANOVA should be considered accurate. Specifically, (sphericity Assumed, time) P < 0.05, while (time * group) P < 0.05, indicating differences in weight changes at different time points. This suggests that the modeling drugs affected weight, and there was an interaction between time and grouping. Between-group comparison: The ANOVA results for different treatment drug groups showed P = 0.199 (>0.05), indicating no significant difference in weight changes between different drug treatments.

[0070] Analysis of covariance was performed on the data before and after modeling. The results showed that after controlling for initial weight, different medications had an impact on weight (P = 0.003), indicating a statistically significant difference. Pairwise comparisons were performed among the four groups: the control group compared with the L-high-citrulline group (Hcit) had a P = 0.001; the control group compared with the behenicol group (Doc) had a P = 0.001; and the control group compared with the L-high-citrulline + behenicol combined treatment group (Doc + Hcit) had a P = 0.007. Figure 2 ).

[0071] Example 3. Observation results of estrous cycles in modeled and treated mice

[0072] During the estrous cycle in mice, a series of histological and physiological changes occur in the reproductive and endocrine systems. Based on these changes, the estrous cycle can be divided into proestrus (P), estrus (E), metestrus (M), and diaestrus (D).

[0073] Based on the results of vaginal smear cytology observation over 14 consecutive days, the estrous cycle of mice was plotted, and the results are shown in Figure 3. Figure 3A The diagram shows the estrous cycle of mice in Example 1. The estrous cycle of mice is approximately 4–5 days, alternating between four phases. If the plotted estrous cycle graph shows an "N", it indicates that the estrous cycle is regular or has returned to normal. Results show:

[0074] In the control group, 4 mice had cessation of estrous cycle, while 3 mice had prolonged estrous cycle.

[0075] In the L-high citrulline treatment group, 1 mouse had estrous cycle arrest, 3 mice had prolonged estrous cycles, and 2 mice had disordered estrous cycles.

[0076] In the behenic acid treatment group, 2 mice had normal estrous cycles, 2 mice had prolonged estrous cycles, and 2 mice had stopped estrous cycles.

[0077] In the L-high-citrulline + behenicol combination therapy group, the estrous cycle was prolonged in 5 mice and the estrous cycle was stopped in 1 mouse. Figure 3A ).

[0078] The proportion of each stage of the estrous cycle in mice of each group during the drug administration period was statistically analyzed, and the results are shown in the figure. Figure 3B middle.

[0079] Statistical analysis was performed on the four stages (P, E, M, and D) of the four groups of mice. No statistically significant differences were found among the groups at any of the four stages. Stage P showed normality and homogeneity of variance (P = 0.561 in one-way ANOVA), indicating no statistically significant difference. Stage E also showed normality and homogeneity of variance (P = 0.157 in one-way ANOVA), indicating no statistically significant difference. Stage M showed normality and homogeneity of variance (P = 0.914 in one-way ANOVA), indicating no statistically significant difference. Stage D did not show normality but homogeneity of variance (P = 0.341 in nonparametric tests), indicating no statistically significant difference. Conclusion: The combined administration of behenic acid, L-homocytrulline, and L-homocytrulline with behenic acid prolonged the estrus period.

[0080] Example 4. Effects of behenic acid and L-hocitrulline on the number of oocytes retrieved after ovulation induction in mice with premature ovarian failure.

[0081] This example investigated the effects of behenic acid and L-homocytrulline on ovulation in mice with ovarian insufficiency. A subset of mice treated in Example 1 were observed: control group (n=4), behenic acid group (n=3), holocitrulline group (n=3), and behenic acid + holocitrulline group (n=3). Methods: After superovulation (injection of 10 IU / mouse of pregnant mare serum, followed by injection of 10 IU / mouse of human chorionic gonadotropin 48 hours later), the ampulla of the fallopian tubes was harvested 14–16 hours later, and the number of oocytes retrieved was recorded. The oocyte counts for the four mice in the control group were 0, 0, 0, and 5; the oocyte counts for the behenic acid group were 22, 34, and 30; the oocyte counts for the L-homocytrulline group were 12, 41, and 9; and the oocyte counts for the combined behenic acid and L-homocytrulline treatment group were 24, 32, and 25. The results showed that behenic acid, high-citrulline, or behenic acid + high-citrulline could treat and restore ovulation in mice with ovarian insufficiency. Figure 4 A and Figure 4 B).

[0082] Example 5. Determination of Oxidative Stress Levels

[0083] The oxidative stress hypothesis posits that a decrease in the body's antioxidant capacity weakens its ability to scavenge free radicals, leading to oxidative damage to biomolecules and an imbalance between cellular oxidation and antioxidant functions, resulting in oxidative stress and consequently disease and aging. Recent reports have shown an association between oxidative stress and ovarian aging and POI in primates. Oxygen free radicals act on unsaturated fatty acids in lipids, generating lipid peroxides, including malondialdehyde (MDA). Antioxidant defense systems that scavenge free radicals include superoxide dismutase (SOD) and reduced glutathione (r-glutamyl cysteinyl glycine, GSH), which can bind to peroxides and free radicals to counteract the damage of sulfhydryl groups by oxidants, protecting sulfhydryl-containing proteins and enzymes in cell membranes from damage, and also combating free radical damage to vital organs. Therefore, this study investigated the effects of three therapeutic drug regimens on oxidative stress in mice with ovarian insufficiency. The results are shown in Table 2. Figure 5 middle.

[0084] Table 2. Comparison of oxidative stress indicators after compound treatment

[0085]

[0086] like Figure 5 As shown in Figure A, after treatment, serum malondialdehyde (MDA) levels were measured. Compared with the control group, MDA levels decreased to some extent in the behenicol treatment group, the L-homocycutaine treatment group, and the behenicol + L-homocycutaine combined treatment group, with significant differences between groups (p = 0.026). Pairwise comparisons between groups revealed a difference between the control group and the behenicol + L-homocycutaine combined treatment group (p = 0.003). This indicates that behenicol, L-homocycutaine, and the combined use of behenicol + L-homocycutaine can reduce MDA levels to some extent.

[0087] like Figure 5 As shown in Figure B, after treatment, compared with the control group, the levels of SOD in the behenic acid treatment group, the L-homocycutaine treatment group, and the behenic acid + L-homocycutaine combined treatment group were all increased to some extent.

[0088] like Figure 5 As shown in Figure C, after treatment, compared with the control group, the activity of GSH was increased in the behenic acid treatment group, the L-homocycutaine treatment group, and the behenic acid + L-homocycutaine combined treatment group. Pairwise comparisons between groups revealed a significant difference between the control group and the behenic acid + L-homocycutaine combined treatment group. This indicates that behenic acid, L-homocycutaine, and the combined use of behenic acid + L-homocycutaine can promote the activity of glutathione to some extent.

[0089] Example 6. Detection of follicles at various stages, corpus luteum, and atretic follicles

[0090] Mice in the three treatment groups (L-high citrulline group, behenicol group, and L-high citrulline + behenicol combined treatment group) were sacrificed, and ovarian tissue was harvested. One ovary was fixed in 4% paraformaldehyde, embedded in paraffin, and then serially sectioned (each section was 5 μm, and one section was collected every 5 sections). Five sections containing the largest facet of each ovary were stained with HE. The counts of primordial follicles, primary follicles, secondary follicles, corpus luteum, and atretic follicles were statistically analyzed (Figure 6).

[0091] The results are as follows Figure 6A and Figure 6B As shown, compared with the control group (Vehicle group), the number of follicles at all stages in the ovaries of mice in the behenic acid (Doc) treatment group and the combined behenic acid and high-citrulline (Doc+Hcit) treatment group was significantly increased, the number of corpora lutea was increased, and the number of atretic follicles was decreased; compared with the control group (Vehicle group), the number of follicles at all stages in mice in the L-high-citrulline (Hcit) treatment group was significantly increased, the number of atretic follicles was decreased, and the number of corpora lutea was decreased.

[0092] Example 7. Detection of gene expression

[0093] Mice in three treatment groups (L-high-citrulline group, behenicol group, and L-high-citrulline + behenicol combined treatment group) were sacrificed, and ovarian tissue was harvested. The contralateral ovary was flash-frozen in liquid nitrogen and stored at -80°C, then sent to Annoroad for RNA transcriptional sequencing. Bioinformatics analysis and mapping were performed on the sequencing results. The RNA levels of the three treatment groups were compared with the control group (vehicle group) to identify significantly differentially expressed genes. The intersection of the differentially expressed genes in the three treatment groups was used to obtain the sets of differentially expressed genes that were commonly upregulated or commonly downregulated in the three treatment groups (FC>10, p<0.05). Figure 7 A and Figure 7 B). Then, gene ontology function (GO) enrichment was performed on these differentially expressed gene sets (fold change (FC) > 10, corrected p-value < 0.05). Figure 8 A and Figure 8 B) and signaling pathway (KEGG) enrichment map ( Figure 9 A and Figure 9 B). Additionally, a heatmap analysis of the expression of significantly upregulated or downregulated genes was performed ( Figure 10 ).

[0094] like Figures 7-10As shown, after three treatments (Doc, Hcit, Doc+Hcit), compared with the control group, 2181, 2282, and 2264 genes were upregulated, respectively, with a total of 1149 genes being upregulated in common. Conversely, 3902, 4542, and 3583 genes were downregulated, respectively, with a total of 2346 genes being downregulated in common (FC>2, p-value <0.05 after correction). Among these, 182 genes were significantly upregulated by 10-fold, and 281 genes were significantly downregulated by 10-fold. Functional enrichment analysis revealed upregulation of receptor binding, molecular binding, and ion channel activity, while immunoglobulin binding and immune responses were downregulated. Signal pathway enrichment results showed upregulation of genes involved in oocyte maturation-related cAMP signaling and steroid hormone production pathways, while downregulation of genes related to calcium signaling pathways. The expression of ovarian reserve-related genes such as Amh and Fshr was significantly upregulated; the upregulation of Amh was more pronounced in the combination therapy group (upregulated by 109, 58, and 126 times, respectively, in the three groups). CYP11b1, 17a1, and 19a1, cytochrome P450 (CYP) superfamily members related to estrogen and progesterone production, were all significantly upregulated. Additionally, important ovarian endocrine-related genes inhba and inhbb were also significantly upregulated. The expression of transcription factor Nr5a2 (LRH-1) was significantly upregulated. Conversely, the expression of genes related to de novo methylation synthesis, such as Dnmt3a, was downregulated, as was the immune-related de-methylating factor cxcl5.

[0095] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. The use of behenic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment or prevention of ovarian dysfunction, wherein, The ovarian dysfunction diseases mentioned are selected from early-onset ovarian insufficiency, premature ovarian failure, and decreased ovarian reserve.

2. The application according to claim 1, characterized in that, The behenic acid or its pharmaceutically acceptable salt can restore ovulation.

3. The application according to claim 1 or 2, characterized in that, The ovarian dysfunction is caused by genetic factors, iatrogenic factors, immune factors, environmental factors, advanced physiological age, or other factors.

4. The use of behenicol or a pharmaceutically acceptable salt thereof, in combination with a compound of formula I or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment or prevention of ovarian dysfunction. in, n is 4; the ovarian dysfunction diseases mentioned are selected from early-onset ovarian insufficiency, premature ovarian failure, and decreased ovarian reserve.

5. The application according to claim 4, characterized in that, The behenic acid or a pharmaceutically acceptable salt thereof, when used in combination with the compound shown in Formula I or a pharmaceutically acceptable salt thereof, can restore ovulation.

6. The application according to claim 4, characterized in that, The ovarian dysfunction is caused by genetic factors, iatrogenic factors, immune factors, environmental factors, advanced physiological age, or other factors.

7. The application according to claim 4, characterized in that, The behenic acid or its pharmaceutically acceptable salt, and the compound represented by Formula I or its pharmaceutically acceptable salt, are administered simultaneously or sequentially.

8. The use of the composition in the preparation of a medicament for the prevention or treatment of ovarian disorders, characterized in that, The composition comprises the following active ingredient: behenic acid or a pharmaceutically acceptable salt thereof; and, the compound represented by Formula I or a pharmaceutically acceptable salt thereof, Wherein, n is 4; the ovarian dysfunction diseases are selected from early-onset ovarian insufficiency, premature ovarian failure, and decreased ovarian reserve.

9. The application according to claim 8, characterized in that, The composition further includes pharmaceutically acceptable excipients.

10. The application according to claim 8, characterized in that, Pharmaceutically acceptable excipients include excipients.

11. The application according to claim 8, characterized in that, The composition is in the form of tablets, capsules, solutions, granules, pills, powders, suspensions, or injections.

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