Drug for treating ovarian insufficiency

Abstract

Provided is a drug for treating ovarian insufficiency. In particular, the present invention relates to the use of annexins in the preparation of a pharmaceutical composition used for preventing and / or treating ovarian insufficiency.

Description

A drug for treating ovarian insufficiency Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a medicine for treating ovarian insufficiency. Background Art

[0002] Ovarian insufficiency (POI) refers to ovarian failure in normally developing women before the age of 40, resulting in premature amenorrhea due to causes other than pregnancy, accompanied by decreased serum estrogen levels and increased gonadotropin levels. POI is one of the main causes of fertility problems in women. Its incidence has been increasing annually in recent years, with a younger prevalence. Its etiology is complex, with advanced age, genetics, tumor radiotherapy and chemotherapy, immune disorders, and metabolic diseases all closely associated with its onset. Currently, hormone replacement therapy is the primary clinical treatment, but its long-term use increases the incidence of ovarian cancer, making its application controversial. Therefore, no reliable treatment has yet been developed to restore ovarian function in POI patients.

[0003] Therefore, there is a need in the art to develop a drug that can effectively treat ovarian insufficiency (POI).

[0004] Summary of the Invention

[0005] The object of the present invention is to provide the use of annexin in preparing a pharmaceutical composition for preventing and / or treating ovarian insufficiency.

[0006] In a first aspect, the present invention provides the use of annexin in the preparation of a pharmaceutical composition for preventing and / or treating ovarian insufficiency.

[0007] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0008] In another preferred embodiment, the use includes one or more features selected from the following group:

[0009] (i) Increase estradiol (E2) levels;

[0010] (ii) increasing anti-Mullerian hormone (AMH) levels;

[0011] (iii) reduction of follicle stimulating hormone (FSH) levels; and / or

[0012] (iv) Reduce the ratio of follicle-stimulating hormone to luteinizing hormone (FSH / LH).

[0013] In another preferred embodiment, the use includes one or more features selected from the following group:

[0014] (1) Promote the proliferation of ovarian granulosa cells;

[0015] (2) inhibiting apoptosis of ovarian granulosa cells;

[0016] (3) Promote normalization of egg growth, maturation and ovulation; and / or

[0017] (4) Promote the normalization of menstruation (cycle, blood volume).

[0018] In another preferred embodiment, the causes of ovarian insufficiency are: great mental stress, irregular diet, staying up late, weight loss, chemotherapy, radiotherapy, gynecological inflammation, congenital ovarian incomplete development, or a combination thereof.

[0019] In another preferred embodiment, the cause of ovarian insufficiency is: insufficient estrogen secretion, insufficient progesterone secretion, increased gonadotropin level, ovarian granulosa cell reduction or apoptosis, or a combination thereof.

[0020] In another preferred embodiment, the annexin is human annexin or non-human mammal annexin.

[0021] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0022] In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0023] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0024] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral solutions, and injections.

[0025] In a second aspect, the present invention provides a pharmaceutical composition for treating ovarian insufficiency, characterized in that the composition comprises:

[0026] (a) Annexin;

[0027] (b) another drug selected from the group consisting of progesterone, estradiol valerate, diethylstilbestrol, or a combination thereof; and

[0028] (c) a pharmaceutically acceptable carrier.

[0029] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0030] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0031] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral solutions, and injections.

[0032] In a third aspect, the present invention provides a method for promoting the proliferation of ovarian granulosa cells and / or inhibiting the apoptosis of ovarian granulosa cells, comprising the steps of contacting and culturing ovarian granulosa cells with annexin, thereby promoting the proliferation of ovarian granulosa cells and / or inhibiting the apoptosis of ovarian granulosa cells.

[0033] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0034] In another preferred embodiment, the concentration of the annexin is 1 ng / mL-10 μg / mL, preferably, 100 ng / mL-1 μg / mL.

[0035] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0036] In a fourth aspect, the present invention provides a method for treating premature ovarian failure, comprising the steps of administering annexin or a pharmaceutical composition comprising annexin as an active ingredient to a subject in need thereof.

[0037] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0038] In another preferred embodiment, the subject is a human or a non-human mammal.

[0039] In another preferred embodiment, the non-human mammal includes pigs, cows, sheep, rats, mice or rabbits.

[0040] In another preferred embodiment, the subject is a subject with ovarian insufficiency.

[0041] In another preferred embodiment, the subject is a subject who has received, is receiving, or will receive treatment for ovarian insufficiency.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 shows the protective effect of Annexin A5 on the ovaries of POI mice. DETAILED DESCRIPTION

[0044] After extensive and intensive research, screening, and testing, the present inventors have developed a drug for treating ovarian insufficiency. They unexpectedly discovered for the first time that annexin can be used to prevent and / or treat ovarian insufficiency, with particular effectiveness in regulating hormones related to ovarian function, promoting ovarian granulosa cell proliferation, and inhibiting ovarian granulosa cell apoptosis. This discovery led to the completion of the present invention.

[0045] the term

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0049] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0050] Annexin

[0051] Annexins are a widely distributed class of calcium-dependent phospholipid-binding proteins. Based on molecular phylogenetic and comparative genomic analyses, this protein family has been uniformly designated "annexin" (abbreviated as Anx), followed by capital letters representing species: A, B, C, D, and E, representing vertebrates, invertebrates, fungi and myxomycetes, plants, and protists, respectively. Currently, the Anx A family has 12 members: A1-A11 and A13 (A12 is unassigned).

[0052] Typically, the annexin includes annexin A5. Annexin A5 (AnxA5) is widely distributed in various tissue cells of the body. In vitro studies have found that AnxA5 has multiple functions such as anti-phospholipase, anti-coagulation, and anti-kinase, but its specific in vivo function is not clear.

[0053] The annexins that can be used in the present invention are not particularly limited and can be annexins derived from any organism, preferably from mammals (such as primates), more preferably from humans, monkeys, rats or mice. The annexins of the present invention can also be functional analogs thereof, such as proteins having 50%, 60%, 70%, 75% or more, such as 80%, 85% or more, 90% or more, or even more preferably 95% or 99% or more identity with human annexin; it should be understood that "annexin" includes wild-type or mutant (including truncated) annexins, as long as the mutant annexins retain or maintain the detoxification activity of the wild-type annexins, they can also be used in the present invention. In addition, the annexins can also be multimers of natural annexins, fusion proteins, or chemically modified variants thereof (such as PEG-modified), provided that these variants have the activity of the wild-type annexins.

[0054] The human annexin A5 sequence is as follows:

[0055] use

[0056] The present invention provides the use of annexin in preparing a pharmaceutical composition for preventing and / or treating ovarian insufficiency, and has significant effects in regulating hormones, promoting ovarian granulosa cell proliferation, and / or inhibiting ovarian granulosa cell apoptosis.

[0057] As used herein, ovarian insufficiency (POI) refers to ovarian failure in normally developed women before the age of 40, leading to premature amenorrhea due to reasons other than pregnancy, accompanied by characteristics such as decreased serum estrogen levels and increased gonadotropin levels. It is usually manifested by symptoms of menstrual abnormalities (such as frequent menstruation, oligomenorrhea, amenorrhea or early menopause) and anovulation (which may lead to infertility). The causes of ovarian insufficiency are complex, and common causes include long-term mental stress, irregular diet, staying up late, gynecological inflammation, weight loss, chemotherapy, congenital ovarian incomplete development, etc.

[0058] Granulosa cells of the ovary are located within the follicle cavity of female animals. They can generally be divided into two major types: the cumulus granulosa cells that surround the oocyte and the parietal granulosa cells that line the follicle wall. The cumulus granulosa cells that surround the oocyte expand before ovulation. After ovulation, the parietal granulosa cells undergo luteinization to form granulosa-lutein cells, which then join with theca cells to form the corpus luteum. This process is crucial for maintaining pregnancy. Follicular development and ovulation depend on highly coordinated development between the ovarian granulosa cells and the oocyte, primarily achieved through continuous, complex, and bidirectional signaling between the two cell types. Microvilli form gap junctions between the cumulus granulosa cells and the oocyte, which facilitate the transfer of nutrients, primary metabolites, and certain signaling molecules, promoting their growth and differentiation and achieving highly coordinated development. Furthermore, the oocyte cannot effectively utilize glucose for energy production and must instead utilize carboxylic acids produced by granulosa cell metabolism through oxidative phosphorylation to generate the energy required for development. In summary, ovarian granulosa cells are crucial support cells for oocyte growth, maturation, and ovulation. The experiments of the present invention also prove that the membrane-binding protein can promote the proliferation of ovarian granulosa cells and / or inhibit the apoptosis of ovarian granulosa cells, thereby promoting egg growth, maturation and ovulation, and improving egg quality.

[0059] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. As used herein, "prevention" also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0060] "Treatment" as used herein includes delaying and stopping the progression of a disease, or eliminating the disease, and does not require 100% inhibition, elimination, or reversal. In some embodiments, the acellular adipose extract of the present invention reduces, inhibits, and / or reverses ovarian insufficiency by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the absence of the acellular adipose extract 28 of the present invention.

[0061] Pharmaceutical composition

[0062] The present invention also provides a pharmaceutical composition comprising the above-mentioned annexin as an active ingredient and a pharmaceutically acceptable carrier.

[0063] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gels suitable for human use, of sufficient purity, and with sufficiently low toxicity. "Compatibility" in this context refers to the ability of the components of the composition to be compatible with the active ingredient of the invention, and with each other, without significantly reducing the compound's efficacy. Such carriers include, but are not limited to, saline, buffered solutions, dextrose, water, glycerol, ethanol, and combinations thereof. The dosage form should be compatible with the route of administration.

[0064] The composition according to the present invention can be used for: parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous administration. Can be by the active ingredient of the required amount in a suitable solvent and various other ingredients (if necessary) as listed above merged, then by sterilization to make a sterile injectable solution. It is best to use the above composition in the form of a sterile aqueous solution, the sterile aqueous solution can contain other substances, such as enough salt or glucose, so that the solution and blood are isotonic. If necessary, the aqueous solution is appropriately buffered (preferably pH between 3-9). Standard pharmaceutical techniques well known to those skilled in the art can be utilized to easily prepare suitable pharmaceutical formulations under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight-about 50 mg / kg body weight every day.

[0065] The active ingredient of the present invention can be used alone or together with one or more other drugs for ovarian insufficiency-related diseases, such as estrogen drugs, for example, progesterone, estradiol valerate, diethylstilbestrol, etc.

[0066] The precise amount of active ingredient provided to an individual for a therapeutically effective amount will depend on the mode of administration, the type and severity of the disease and / or condition, and the characteristics of the individual, such as general health, age, sex, weight, and tolerance to the drug. One of ordinary skill in the art will be able to determine an appropriate dosage based on these and other factors. When administered in combination with other therapeutic agents, the "therapeutically effective amount" of any other therapeutic agent will depend on the type of drug used. Suitable dosages are known for approved therapeutic agents and can be adjusted by one of ordinary skill in the art based on the individual's condition, the type of condition being treated, and the amount of the compound of the present invention used below, for example, as reported in the literature and recommended in the Physician's Desk Reference (57th Edition, 2003). Preferably, the composition should be formulated so that an inhibitor dose of 0.01-100 mg / kg body weight / day can be administered to patients receiving these compositions. In certain embodiments, the compositions of the present invention provide a dosage of 0.01 mg to 50 mg. In other embodiments, a dosage of 0.1 mg to 25 mg or 5 mg to 40 mg is provided.

[0067] The present invention also provides a method for treating ovarian insufficiency, comprising the steps of administering a pharmaceutical composition comprising annexin as an active ingredient to a subject in need thereof.

[0068] Examples of subjects to whom the active ingredient of the present invention or a pharmaceutical composition comprising the same can be administered include mammals (eg, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).

[0069] The main advantages of the present invention include:

[0070] The present inventors discovered for the first time that annexin can be used to prevent and / or treat ovarian insufficiency.

[0071] The present inventors discovered for the first time that annexin can regulate hormones related to ovarian function, promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of ovarian granulosa cells.

[0072] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0073] Annexin A5: extracted and purified from human adipose tissue, accession number: P08758:

[0074] Example 1

[0075] 1. Animal Experiment Content

[0076] 1.1 Model preparation

[0077] Female C57 mice, 4-6 weeks old, were selected and divided into experimental and control groups, with 15 mice in the experimental group and 10 mice in the control group. The experimental group received a single intraperitoneal injection of cyclophosphamide (120 mg / kg) and an intradermal injection of busulfan (12 mg / kg, totaling 0.1 mL) in PBS to establish a POI model; the control group was housed normally. Two weeks later, samples were collected from five mice in the experimental group for hormone levels (AMH, E2, and FSH), as well as ovarian histology, to confirm successful establishment of the POI model.

[0078] 1.2 Drug administration experiment

[0079] After confirming the successful establishment of the POI mouse model, the experimental group of mice was treated with Annexin A5. Annexin A5 was administered intraperitoneally at a concentration of 1 mg / ml, with 0.1 ml administered twice weekly. The experimental group of mice was sacrificed 14 and 28 days after treatment, and ovaries were harvested from five mice. The same number of ovaries were harvested from the control group at the same time for hormone analysis, including AMH, E2, and FSH levels, as well as ovarian histology.

[0080] The experimental results showed that (1) the levels of E2 (estradiol) and AMH (anti-Mullerian hormone) in POI mice decreased significantly. Two weeks after intraperitoneal injection of annexin A5, the E2 hormone level increased significantly and remained stable until the four-week observation endpoint. The difference in hormone levels before and after injection of annexin A5 was statistically significant. (2) The experimental results also showed that the FSH (follicle-stimulating hormone) hormone level in mice with ovarian insufficiency caused by chemotherapy increased significantly. After intraperitoneal injection of annexin A5, the FSH value decreased significantly, and at the four-week observation endpoint, the value continued to decrease and gradually stabilized. The difference in hormone levels before and after injection of annexin A5 was statistically significant.

[0081] The results showed that annexin A5 significantly increased the levels of E2 and AMH hormones in POI mice and significantly decreased the levels of FSH hormones, which was beneficial to the recovery of ovarian function (*P<0.05). This was confirmed by the ovarian histological results. As shown in Figure 1, the ovarian tissue structure of the control group (Control) was basically normal, with a complete follicular structure visible in the field of view, clear oocyte structure, rich cytoplasm, and eosinophilicity; the granulosa cell nuclei were round, abundant, and neatly arranged, with no necrosis; no obvious inflammatory cell infiltration was observed in the interstitium; the ovarian tissue structure of the POI model group (Model) was mild to moderately abnormal, with a small amount of granulosa cell necrosis and deep nuclear condensation in the follicles. The ovarian tissue structure of the experimental group (AnxA5) was basically normal, with complete follicular structure, clear oocyte structure, abundant cytoplasm and eosinophilicity; the granulosa cells had round nuclei, abundant number, and were arranged neatly and tightly, without necrosis; the volume of the mesenchymal cells in the interstitial glands increased, the cytoplasm was filled with lipid droplets, and the connective tissue and blood vessels were separated into cell clusters and cords. The purple arrows indicate the interstitial glands; there was no obvious inflammatory cell infiltration in the interstitium, indicating that annexin A5 has a significant protective effect on the ovarian function of POI mice.

[0082] Example 2

[0083] Cell experiments

[0084] 1.2 Experimental cell treatment

[0085] Primary human ovarian granulosa cells (KGN) were purchased from Shanghai Yuka Biotechnology Co., Ltd. and cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% double-antibody. Cells were passaged when the cell density reached 90%. KGN cells were treated with 250 μM cyclophosphamide (CTX) for 48 hours to induce injury, and cell viability was measured.

[0086] 1.3 Drug treatment

[0087] 2.2.1 Effect of Annexin A5 on the Proliferation Ability of KGN-CTX

[0088] After cell digestion, a cell suspension was prepared and 2000 cells / well were seeded into a 96-well plate and cultured in a 37°C incubator for 24 hours. The next day, the 96-well plate cells were treated with different concentrations of annexin A5, with the concentrations set as: 0, 1, 10, 100, and 1000 ng / ml, with 4 replicates per group, and cultured for another 72 hours. After 72 hours of drug incubation, the 96-well plate was removed, 10ul of CCK8 solution was added to each well of cells, mixed, and incubated in a 37°C incubator for 1 hour. The cells were then detected using a microplate reader and the proliferation capacity of each group of cells was calculated.

[0089] 2.2.2 Effect of Annexin A5 on the anti-apoptotic ability of KGN-CTX

[0090] The anti-apoptotic capacity of normal KGN cells, KGN-CTX cells, and KGN-CTX cells treated with various concentrations of Annexin A5 (0, 1, 10, 100, and 1000 ng / mL) was assessed using a TUNEL apoptosis detection kit (Beyotime). Following the manufacturer's instructions, cells were harvested and incubated with TUNEL assay solution to label the nuclei with Hoechst 33342. Samples were analyzed using a fluorescence microscope.

[0091] The experimental results showed that annexin A5 can significantly promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of ovarian granulosa cells.

[0092] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of annexin in the preparation of a pharmaceutical composition for preventing and / or treating ovarian insufficiency.

2. The use according to claim 1, characterized in that The annexin comprises annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

3. The use according to claim 1, characterized in that The use comprises one or more features selected from the group consisting of: (i) Increase estradiol (E2) levels; (ii) Increase anti-Mullerian hormone (AMH) levels; (iii) reducing anti-follicle stimulating hormone (FSH) levels; and / or (iv) Decrease the ratio of follicle stimulating hormone to luteinizing hormone (FSH / LH).

4. The use according to claim 1, characterized in that The use comprises one or more features selected from the group consisting of: (1) Promote the proliferation of ovarian granulosa cells; (2) inhibiting apoptosis of ovarian granulosa cells; (3) Promote normalization of oocyte growth, maturation and ovulation; and / or (4) Promote normalization of menstruation (cycle, blood volume).

5. The use according to claim 1, characterized in that The causes of ovarian insufficiency are: insufficient estrogen secretion, insufficient progesterone secretion, increased gonadotropin levels, reduction or apoptosis of ovarian granulosa cells, or a combination thereof.

6. The use according to claim 1, characterized in that The causes of ovarian insufficiency are: great mental stress, irregular diet, staying up late, weight loss, chemotherapy, radiotherapy, gynecological inflammation, congenital ovarian incomplete development, or a combination thereof.

7. The use according to claim 1, characterized in that The annexin is human annexin or non-human mammal annexin.

8. The use according to claim 7, characterized in that The annexin is human annexin A5.

9. The use according to claim 1, characterized in that The composition also includes a pharmaceutically acceptable carrier.

10. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

11. The use according to claim 1, characterized in that The dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral liquids, and injections.

12. A pharmaceutical composition for treating ovarian insufficiency, characterized in that: The composition comprises: (a) Annexin; (b) another drug selected from the group consisting of progesterone, estradiol valerate, ethinylestradiol, or a combination thereof; and (c) a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, characterized in that The annexin includes annexin A5.

14. The pharmaceutical composition according to claim 12, characterized in that The dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral liquids, and injections.

15. A method for promoting proliferation of ovarian granulosa cells and / or inhibiting apoptosis of ovarian granulosa cells, comprising the steps of: contacting ovarian granulosa cells with annexin and culturing them, thereby promoting proliferation of ovarian granulosa cells and / or inhibiting apoptosis of ovarian granulosa cells.

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