Application of DON in relieving ovarian fibrosis

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of DON in relieving ovarian fibrosis. The invention discloses an application of DON or a pharmaceutically acceptable derivative thereof in preparation of a medicine for treating or preventing ovarian fibrosis. The invention discloses application of DON in prevention and treatment of ovarian fibrosis, human ovarian is cultured in vitro, DON is added for treatment, and the result shows that DON can reduce the ovarian fibrosis level, so that DON can be used as a potential medicine for recovering female fertility. The DON is prompted to have an obvious promoting effect on recovery of ovarian fibrosis, so that the DON can be used for solving the problems of ovarian fibrosis, non-ovulation and the like caused by endometriosis, and a new medicine choice is provided for clinical treatment of infertility.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of DON in alleviating ovarian fibrosis. Background Technology

[0002] The ovary is the primary reproductive organ of female mammals, responsible for producing eggs and secreting sex hormones. The normal functioning of the ovaries directly affects the fertility and reproductive health of female animals. Endometriosis is a common gynecological disease, prevalent in women aged 25 to 45, with the ovarian type being the most common. Ovarian endometriosis (hereinafter referred to as endometriosis) leads to a significant increase in ovarian fibrosis, causing difficulty in ovulation and clinically manifesting as infertility.

[0003] 6-Diazo-5-oxonorleucine (DON, CAS No. 157-03-9) is a glutamine antagonist that irreversibly inhibits the breakdown of glutamine. Studies have shown that DON has good anticancer activity (especially in pancreatic cancer), reducing the self-renewal potential and metastatic ability of tumor cells. DON also has antibacterial and antiviral activity. However, there are currently no clear reports on the application of DON in the recovery of ovarian endometriosis fibrosis.

[0004] Therefore, in order to solve the above problems, developing a drug-based treatment for infertility caused by ovarian endometriosis fibrosis is of great significance to clinical medicine and infertile patients. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. This invention proposes the application of DON in alleviating ovarian fibrosis, which can achieve the technical effect of alleviating ovarian fibrosis based on drug therapy, and has extremely high clinical application value.

[0006] The first aspect of the present invention aims to provide the use of DON or a pharmaceutically acceptable derivative thereof in the preparation of medicaments for the treatment or prevention of ovarian fibrosis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the invention provides the use of DON or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for the treatment or prevention of ovarian fibrosis.

[0009] In some embodiments of the present invention, the pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and eutectics of DON.

[0010] In some embodiments of the present invention, the pharmaceutical salt of the DON includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0011] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0012] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0013] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0014] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0015] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0016] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0017] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0018] In some embodiments of the present invention, the salt formed with the acidic amino acid includes at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0019] In some embodiments of the present invention, the effective dose of the DON or its pharmaceutically acceptable derivative for the treatment or prevention of ovarian fibrosis is 30-70 μM, such as any value or range formed by any combination of 30, 35, 40, 45, 50, 55, 60, 65 or 70 μM.

[0020] In some embodiments of the present invention, the ovarian fibrosis includes ovarian fibrosis caused by endometriosis.

[0021] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0022] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from at least one of fillers, binders, disintegrants, lubricants, flavoring agents, colorants, masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers.

[0023] In some embodiments of the present invention, for ease of administration, active DON or its pharmaceutically acceptable salts can be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic regulators (such as sodium chloride and glucose).

[0024] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0025] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0026] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0027] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0028] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0029] The beneficial effects of this invention are: This invention discloses the application of DON in the prevention and treatment of ovarian fibrosis. The invention utilizes in vitro cultured human ovaries, treated with added DON. Results show that DON can alleviate ovarian fibrosis levels, thus serving as a potential drug for restoring female fertility. This suggests that DON has a significant promoting effect on the recovery of ovarian fibrosis, and therefore can be used for ovarian fibrosis and anovulation caused by endometriosis, providing a new drug option for the clinical treatment of infertility. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of fibrosis under polarized light after treating the ovaries of endometriosis patients with 50 μM DON are shown. The scale bar is 20 μM.

[0031] Figure 2 Statistical results of fibrosis under polarized light after treating the ovaries of endometriosis patients with 50 μM DON.

[0032] Figure 3 Statistical results of fibrosis under polarized light after treating the ovaries of endometriosis patients with 25 μM DON.

[0033] Figure 4 The mouse's condition during animal toxicology testing.

[0034] Figure 5 Flow cytometry results for cell state in cytotoxicity assays. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] The term "ovarian fibrosis" refers to the numerous anatomical and physiological changes that occur in women during each ovarian cycle, including follicular growth (follicle development), hormone production, ovulation, and stromal remodeling. Normal follicle development is accompanied by an increase in pro-inflammatory cytokines, which are involved in the ability to acquire oocytes for development during follicular growth and allow for stromal remodeling of the ovarian stroma to weaken the ovarian wall after follicle rupture and participate in wound repair after ovulation. Chronic inflammation is associated with the development of fibrosis in other tissues, and most chronic fibrotic diseases have persistent irritants that enhance the deposition of fibrotic cytokines and connective tissue, thereby altering normal tissue. In the case of the ovary, the recurring inflammatory process of ovulation and wound healing can be a persistent irritant, leading to age-related ovarian fibrosis and higher levels of permanent collagen deposition. Severe ovarian fibrosis can lead to impaired ovulation and damage ovarian function.

[0038] The term "endometriosis" refers to a common gynecological condition that predominantly affects women aged 25 to 45, with the ovarian type being the most common. Ovarian endometriosis leads to a significant increase in ovarian fibrosis, causing difficulty in ovulation and clinically manifesting as infertility.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1: The effect of DON on the recovery of endometriosis or ovarian fibrosis Experimental materials (1) Ovarian tissue from patients with endometriosis: Human ovarian samples were collected from patients with endometriosis (21-34 years old) at Nanfang Hospital. Before surgery, they were diagnosed with grade III and IV ovarian endometriosis according to the guidelines of the American Society for Reproductive Medicine via laparoscopy. Patients with endometriosis underwent oophorectomy, and ectopic lesions on the ovaries were removed postoperatively.

[0041] (2) DON: The CAS number of DON is 157-03-9, the compound name is 6-Diazo-5-oxo-L-norleucine, purchased from Selleck, and the batch number is S862001.

[0042] The DON used includes DON and DON analogues.

[0043] To prepare the DON solution for in vitro culture, take 4.279 mg of DON powder and dissolve it in 1 mL of ddH2O as a solvent under ultrasonic or 37°C conditions to obtain a stock solution with a final concentration of 25 mM. Store the stock solution at -20°C or -80°C. Dilute to the required concentration with culture medium before use.

[0044] Experimental methods (1) The lesions dissected from four laparoscopic surgery patients were placed in tissue preservation solution (Medtronic Biotechnology Co., Ltd., 130-100-008) and transported to the laboratory on ice. On ice, the ovarian tissue was washed in PBS, and the ovarian medulla and lesion sites were removed with a scalpel, while the normal ovarian cortex was preserved.

[0045] (2) Ovarian tissues from four endometriosis patients were transferred to DMEM / F12 culture medium and minced with scissors to obtain tissues with a size of 1 mm. 3 Tissue blocks were randomly and evenly placed into six-well plates containing 3 mL of culture medium and Millicell inserts. 20-30 tissue blocks were added to each well (four droplets per well, five tissue blocks per droplet, 20 μL volume per droplet, one six-well plate per patient).

[0046] (3) For each patient, DON was added to the culture medium in wells 1-3 to a final concentration of 25 μM or 50 μM, and an equal volume of ddH2O was added to wells 4-6. The mixture was incubated at 37°C and 5% CO2 for two days. DON was dissolved in ddH2O to a 50 mM concentrated stock solution for storage.

[0047] (4) After the cultured tissues were divided into groups, they were placed into 1.5 mL ep tubes containing 1 mL of 4% paraformaldehyde solution for fixation (16 h). The tissues from each well were added to the same tube.

[0048] (5) Tissue embedding: Ovarian tissue was placed in an embedding cassette and marked with a pencil. It was then sequentially immersed in 70% ethanol solution, 80% ethanol solution, 90% ethanol solution, 100% ethanol solution I, and 100% ethanol solution II for one hour each. Next, it was sequentially immersed in xylene I, xylene II, and paraffin xylene 1:1 (volume ratio) solution for 0.5 hours. Finally, it was sequentially immersed in paraffin I and paraffin II for 2 hours each. The tissue was then embedded using a Zeiss embedding instrument. A total of 120 small tissue pieces were embedded from the ovarian tissues of four patients, with 10 small tissue pieces encapsulated in one paraffin block. Each patient had 6 paraffin blocks in the control group and 6 paraffin blocks in the DON treatment group.

[0049] (6) Tissue sectioning: The paraffin tissue was sectioned using Zeiss paraffin sectioning blades and Zeiss microtome to a thickness of 5 μm.

[0050] (7) PSR staining: Ovarian tissue sections were stained with PSR staining solution (Solebo, G1472) and then observed and photographed under a microscope.

[0051] (8) Data statistics: The area of ​​ovarian fibrosis was calculated using imagej software based on the PSR staining results.

[0052] Statistical analysis of ovarian fibrosis area shows that after treatment with 50μM DON, the fibrosis area of ​​ovarian tissue in endometriosis patients decreased, and the level of visible fibrosis was significantly reduced. Figure 1 Compared with the control group, the fibrosis level in the treatment group (50 μM DON treatment) decreased by 19.10%. Figure 2 Each point represents one patient), and there was no significant difference in fibrosis levels between the 25 μM DON administration group and the control group (where each point represents one patient). Figure 3 This suggests that DON can slow down ovarian fibrosis within a specific dose range and can be used to treat ovarian fibrosis.

[0053] Example 2: Safety Testing of DON This embodiment examines the safety of DON in the treatment of ovarian fibrosis through animal and cytotoxicological tests, as detailed below: Animal toxicology test: Three 8-week-old C57 mice were intraperitoneally injected with 0.5 mM DON. 100 μL was administered per 100 g body weight, and the mice were observed at 12 h, 24 h, and 48 h after injection.

[0054] Cytotoxicity assay: Using the KGN cell line, when the cell density reached 70%, 50 μM DON was added to DMEM / F12 medium and treated at 37°C and 5% CO2 for 24 h. Cells were then collected, stained with dapi for 15 min, and flow cytometry was performed to detect the number of dapi-positive cells.

[0055] Animal toxicology test results as follows Figure 4 As shown, the c57 mice were in good condition and showed no abnormalities for a period of time after intraperitoneal injection of DON, suggesting that DON has good safety.

[0056] Cytotoxicology test results as follows Figure 5 As shown, there was no significant difference in the proportion of dapi-positive cells between the experimental group and the control group (which had the same volume of solvent as the experimental group: ddH2O added). 50 μM DON had no significant effect on cell viability, suggesting that DON has good safety.

[0057] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The use of DON or pharmaceutically acceptable derivatives thereof in the preparation of medicaments for the treatment or prevention of ovarian fibrosis.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable derivatives include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, glycosides, solvates, and cocrystals of DON.

3. The application according to claim 2, characterized in that, The pharmaceutical salts of DON include at least one of the following: metal salts, ammonium salts, salts formed with inorganic acids, salts formed with organic bases, salts formed with organic acids, salts formed with basic amino acids, and salts formed with acidic amino acids.

4. The application according to claim 3, characterized in that, The metal salt includes alkali metal salts and alkaline earth metal salts; and / or, the alkali metal salt includes at least one of sodium salts and potassium salts; and / or, the salt formed with the inorganic acid includes salts formed with at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

5. The application according to claim 3, characterized in that, The salts formed with organic acids include salts formed with at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

6. The application according to claim 3, characterized in that, The salt formed with a basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, ornithine; and / or, the salt formed with an acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

7. The application according to any one of claims 1-6, characterized in that, The effective dose of DON or its pharmaceutically acceptable derivatives for the treatment or prevention of ovarian fibrosis is 30-70 μM.

8. The application according to any one of claims 1-6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following: fillers, binders, disintegrants, lubricants, flavoring agents, colorants, flavor masking agents, pH adjusters, buffers, preservatives, stabilizers, antioxidants, wetting agents, humidity regulators, surfactants, suspending agents, and absorption enhancers.

10. The application according to any one of claims 1-6, characterized in that, The ovarian fibrosis includes ovarian fibrosis caused by endometriosis.

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