Medicine for treating ovarian insufficiency

Abstract

The invention provides a medicine for treating ovarian insufficiency. In particular to application of annexin in preparation of a pharmaceutical composition for preventing and / or treating ovarian insufficiency.

Description

Technical Field

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

[0002] Ovarian insufficiency (POI) refers to ovarian failure in normally developed women before the age of 40, which leads to premature amenorrhea due to reasons other than pregnancy, accompanied by decreased serum estrogen levels and increased gonadotropin levels. POI is one of the main causes affecting female fertility. In recent years, the incidence rate has increased year by year and tends to be younger. Its etiology is relatively complex. Advanced age, genetics, tumor radiotherapy and chemotherapy, immune disorders, metabolic diseases, etc. are closely related to the onset of the disease. At present, hormone replacement therapy is mainly used in clinical practice, but long-term use leads to an increase in the incidence of ovarian cancer, making the application of this therapy controversial. Therefore, there is no reliable therapeutic drug that can help POI patients restore ovarian function.

[0003] Therefore, there is a need in the art to develop a drug that is effective in treating ovarian insufficiency (POI). Summary of the invention

[0004] 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.

[0005] 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.

[0006] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0007] In another preferred embodiment, the use comprises one or more features selected from the following group:

[0008] (i) Increase estradiol (E2) levels;

[0009] (ii) Increase anti-Mullerian hormone (AMH) levels;

[0010] (iii) reducing anti-follicle stimulating hormone (FSH) levels; and / or

[0011] (iv) Decrease the ratio of follicle stimulating hormone to luteinizing hormone (FSH / LH).

[0012] In another preferred embodiment, the use comprises one or more features selected from the following group:

[0013] (1) Promote the proliferation of ovarian granulosa cells;

[0014] (2) inhibiting apoptosis of ovarian granulosa cells;

[0015] (3) Promote normalization of oocyte growth, maturation and ovulation; and / or

[0016] (4) Promote normalization of menstruation (cycle, blood volume).

[0017] 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.

[0018] In another preferred embodiment, the cause of ovarian insufficiency is: insufficient estrogen secretion, insufficient progesterone secretion, increased gonadotropin level, reduction or apoptosis of ovarian granulosa cells, or a combination thereof.

[0019] In another preferred embodiment, the annexin is human annexin or non-human mammal annexin.

[0020] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0021] In another preferred embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0022] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0023] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral liquids, and injections.

[0024] In a second aspect, the present invention provides a pharmaceutical composition for treating ovarian insufficiency, characterized in that the composition comprises:

[0025] (a) Annexin;

[0026] (b) another drug selected from the group consisting of progesterone, estradiol valerate, diethylstilbestrol, or a combination thereof; and

[0027] (c) a pharmaceutically acceptable carrier.

[0028] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0029] In another preferred embodiment, the dosage form of the composition is selected from the following group: liquid dosage form, solid dosage form.

[0030] In another preferred embodiment, the dosage form of the composition is selected from the following group: tablets, capsules, microcapsules, powder injections, oral liquids, and injections.

[0031] The third aspect of 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.

[0032] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0033] In another preferred embodiment, the concentration of the annexin is 1 ng / mL-10 μg / mL, preferably, 100 ng / mL-1 μg / mL.

[0034] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0035] 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.

[0036] In another preferred embodiment, the annexin includes annexin A5. Preferably, annexin A5 accounts for 80%, 85%, 90%, 95%, 98% or 99% of the annexin.

[0037] In another preferred embodiment, the subject is a human or a non-human mammal.

[0038] In another preferred embodiment, the non-human mammal includes pigs, cows, sheep, rats, mice or rabbits.

[0039] In another preferred embodiment, the subject is a subject with ovarian insufficiency.

[0040] In another preferred embodiment, the subject is a subject who has received, is receiving, or will receive treatment for ovarian insufficiency.

[0041] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0042] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a drug for treating ovarian insufficiency. The inventors unexpectedly discovered for the first time that annexin can be used to prevent and / or treat ovarian insufficiency, especially has significant effects in regulating ovarian function-related hormones, promoting ovarian granulosa cell proliferation, and inhibiting ovarian granulosa cell apoptosis. The present invention was completed on this basis.

[0043] the term

[0044] 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.

[0045] As used herein, when used in reference to a specific recited numerical 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.).

[0046] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0047] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0048] Annexin

[0049] Annexins are a class of widely distributed calcium-dependent phospholipid-binding proteins. Based on molecular phylogenetics and comparative genomics analysis, this protein family is uniformly named annexin (abbreviated as Anx), followed by capital letters to represent different species: A, B, C, D, and E represent vertebrates, invertebrates, fungi and slime molds, plants, and protists, respectively. So far, the Anx A family has 12 members: A1-A11 and A13 (A12 is unspecified).

[0050] 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 very clear.

[0051] 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 wild-type annexins, they can also be used in the present invention. In addition, the annexins can also be polymers of natural annexins, fusion proteins, or chemically modified variants thereof (such as PEG modification), provided that these variants have the activity of wild-type annexins.

[0052] The human annexin A5 sequence is as follows:

[0053] MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRD PDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD(SEQ ID No:1)

[0054] use

[0055] The present invention provides the use of annexin in preparing a pharmaceutical composition for preventing and / or treating ovarian insufficiency, and has a significant effect in regulating hormones, promoting ovarian granulosa cell proliferation and / or inhibiting ovarian granulosa cell apoptosis.

[0056] As used in the present invention, ovarian insufficiency (POI) refers to ovarian failure in normally developed women before the age of 40, which leads 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 as symptoms of abnormal menstruation (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, radiotherapy and chemotherapy, and congenital incomplete ovarian development.

[0057] Ovarian granulosa cells exist in the follicular cavity of female animals. Generally, granulosa cells can be divided into two categories: one is the cumulus granulosa cells wrapped around the oocyte, and the other is the parietal granulosa cells arranged on the wall of the follicle. The cumulus granulosa cells that wrap around the oocyte will spread before ovulation. After ovulation, the parietal granulosa cells will undergo luteinization to form granulosa-lutein cells, and then form a corpus luteum with theca cells. This process is crucial to maintaining pregnancy. The development of follicles and ovulation depend on the highly coordinated development between ovarian granulosa cells and oocytes, and this highly coordinated development is mainly achieved through continuous, complex, and bidirectional signal exchange between the two cells. The gap junctions between the cumulus granulosa cells and the oocytes are formed through microvilli. The two cells complete the transmission of nutrients, primary metabolites and some information molecules through gap junctions, thereby promoting the growth and differentiation of the two cells, so that the two achieve highly coordinated development. In addition, the oocyte cannot effectively use glucose to generate energy, and can only use the carboxylic acids produced by the metabolism of granulosa cells to produce the energy required for development through oxidative phosphorylation reactions. In short, ovarian granulosa cells are important supporting cells that promote egg growth, maturation and ovulation. The experiments of the present invention also prove that the annexin can promote the proliferation of ovarian granulosa cells and / or inhibit the apoptosis of ovarian granulosa cells, thereby promoting the growth, maturation and ovulation of eggs and improving the quality of eggs.

[0058] In the present invention, the term "prevention" means a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0059] The "treatment" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, compared with the absence of the cell-free fat extract 28 described in the present invention, the cell-free fat extract described in the present invention reduces, inhibits and / or reverses ovarian insufficiency, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0060] Pharmaceutical composition

[0061] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned annexin as an active ingredient and a pharmaceutically acceptable carrier.

[0062] The term "pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the compound. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The dosage form of the drug should match the mode of administration.

[0063] Compositions according to the present invention can be used for: parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular or subcutaneous administration. A sterile injectable solution can be prepared by merging the required amount of active ingredient in a suitable solvent with various other ingredients listed above (if necessary), and then sterilizing. It is best to use the above composition in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salt or glucose, to make the solution isotonic with blood. If necessary, the aqueous solution is appropriately buffered (preferably pH between 3-9). Suitable pharmaceutical formulations can be easily prepared under aseptic conditions using standard pharmaceutical techniques well known to those skilled in the art. 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 per day.

[0064] The active ingredient of the present invention can be used alone or together with one or more other drugs for diseases related to ovarian insufficiency, such as estrogen drugs, for example, progesterone, estradiol valerate, ethinyl estradiol, and the like.

[0065] 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, body weight, and tolerance to the drug. One of ordinary skill in the art will be able to determine the 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 composition of the present invention provides a dosage of 0.01 mg to 50 mg. In other embodiments, a dosage of 0.1 mg-25 mg or 5 mg-40 mg is provided.

[0066] 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.

[0067] Examples of subjects to which 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.).

[0068] The main advantages of the present invention include:

[0069] The present inventors have discovered for the first time that annexin can be used to prevent and / or treat ovarian insufficiency.

[0070] The inventors first discovered that annexin can regulate hormones related to ovarian function, promote the proliferation of ovarian granulosa cells, and inhibit the apoptosis of ovarian granulosa cells.

[0071] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0072] Annexin A5: extracted and purified from human adipose tissue, accession number: P08758:

[0073] Example 1

[0074] 1. Animal Experiment Content

[0075] 1.1 Model preparation

[0076] Female C57 mice, 4-6 weeks old, were selected and set up in 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 loading dose of 120 mg / kg combined with intradermal injection of busulfan 12 mg / kg, totaling 0.1 mL (with PBS as solvent) to establish the POI model; the control group was raised normally. After 2 weeks, samples were collected from 5 mice in the experimental group, and hormones including AMH (anti-Muller test tube hormone), E2 (estradiol), FSH (follicle stimulating hormone) levels and ovarian histology were tested to determine that the ovarian insufficiency (POI) model was successfully established.

[0077] 1.2 Drug administration experiment

[0078] After confirming that the POI mouse model was successfully established, mice in the experimental group were treated with annexin A5. The concentration of annexin A5 was adjusted to 1 mg / ml for intraperitoneal injection, and the drug was administered twice a week, 0.1 ml each time. The mice in the experimental group were killed on days 14 and 28 after treatment, and the ovaries of 5 mice were sampled. The same number of samples were collected from mice in the control group at the same time, and hormones including AMH, E2, FSH levels and ovarian histology were tested.

[0079] 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.

[0080] 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), and this was confirmed by the ovarian histological results.

[0081] Example 2

[0082] Cell experiments

[0083] 1.2 Experimental cell treatment

[0084] Human primary ovarian granulosa cells (KGN) were purchased from Shanghai Yuka Biotechnology Co., Ltd. and cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody. Cells were passaged when the cell density reached 90%. 250uM cyclophosphamide (CTX) was added to the culture medium, and KGN cells were induced to damage for 48h, and cell activity was detected.

[0085] 1.3 Drug treatment

[0086] 2.2.1 Effect of Annexin A5 on the proliferation ability of KGN-CTX:

[0087] After the cells were digested, a cell suspension was prepared and inoculated into a 96-well plate at a volume of 2000 cells / well, 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 to: 0, 1, 10, 100, 1000 ng / ml, 4 replicates per group, and continued to be cultured for 72 hours. After 72 hours of drug incubation, the 96-well plate was removed, 10ul CCK8 solution was added to each well of cells, mixed and incubated in a 37°C incubator for 1 hour, then detected using an ELISA instrument, and the proliferation capacity of each group of cells was calculated.

[0088] 2.2.2 Effect of annexin A5 on the anti-apoptotic ability of KGN-CTX

[0089] The anti-apoptosis ability of normal KGN cells, KGN-CTX cells and KGN-CTX cells treated with different concentrations of annexin A5 (the concentrations were set to: 0, 1, 10, 100, 1000 ng / mL) was tested using the TUNEL cell apoptosis detection kit (Biyuntian). According to the manufacturer's instructions, the cells were collected and incubated with the TUNEL detection solution to label the cell nuclei with Hoechst33342, and the samples were analyzed by fluorescence microscopy.

[0090] The experimental results showed that annexin A5 can significantly promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of ovarian granulosa cells.

[0091] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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) increasing anti-Mullerian hormone (AMH) levels; (iii) reduction of follicle stimulating hormone (FSH) levels; and / or (iv) Reduce the ratio of follicle-stimulating hormone to luteinizing hormone (FSH / LH).

4. The use according to claim 1, wherein 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 egg growth, maturation and ovulation; and / or (4) Promote the 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, ovarian granulosa cell reduction or apoptosis, or a combination thereof.

6. The use according to claim 1, wherein The annexin is human annexin or non-human mammal annexin.

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

8. 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.

9. 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, diethylstilbestrol, or a combination thereof; and (c) a pharmaceutically acceptable carrier.

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