Application of β-hydroxybutyric acid in the preparation of drugs for treating endometriosis

Abstract

This invention belongs to the field of biomedicine and specifically relates to the use of β-hydroxybutyrate in the preparation of a drug for the treatment of endometriosis. In animal experiments, BHB treatment significantly reduced endometriosis lesions between days 4 to 18 and 28 after modeling. Clinical studies have also found decreased serum BHB levels in endometriosis patients, suggesting that BHB supplementation may improve endometriosis symptoms. This suggests that BHB has a beneficial therapeutic effect on endometriosis and holds promise for clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of beta-hydroxybutyric acid in the preparation of a drug for treating endometriosis. Background Art

[0002] Endometriosis (ENDOMETRIOSIS) is a common chronic endometrial disease characterized by the presence of endometrial tissue (glands and stroma) outside the uterine corpus. The incidence of endometriosis is approximately 10% to 15% in women of childbearing age, while it reaches 25% to 50% in infertile women, with an annual increase. Although a benign disease, it has a high recurrence rate and is difficult to cure, severely impacting patients' physical and mental health and quality of life. Some patients may even experience a range of psychiatric symptoms, such as depression and anxiety, which negatively impact their daily lives, sexual function, and interpersonal relationships.

[0003] The etiology and pathogenesis of endometriosis remain incompletely understood, posing a challenge to its clinical treatment. Among existing theories, the most widely accepted is the retrograde menstrual implantation theory proposed by Sampson. This theory posits that during menstruation, some menstrual blood containing active endometrial cells retrogradely flows through the fallopian tubes into the pelvic cavity. These endometrial cells then implant and grow in sites such as the peritoneum and ovaries, ultimately forming ectopic lesions. In addition to the retrograde menstrual implantation theory, several other important pathogenesis theories have been proposed. The coelomic metaplasia theory posits that, under specific stimulation, coelomic epithelial cells, such as the pelvic peritoneum and ovarian surface epithelium, undergo metaplastic transformation, differentiating into functional endometrial-like tissue. The induction theory emphasizes that specific inducible factors in the local microenvironment may promote the transformation of eutopic endometrial cells into ectopic endometrial cells. Finally, the immune theory, based on immunoregulatory principles, suggests that abnormal immune system function may prevent the effective recognition and elimination of ectopic endometrial cells, thereby creating conditions for their survival and proliferation. These complementary theories together constitute the current multidimensional framework for understanding the pathogenesis of endometriosis.

[0004] The clinical treatment of endometriosis primarily relies on surgical intervention and drug control. The choice of which strategy should be considered comprehensively, including disease severity, patient age, and fertility needs. Surgical treatment can be divided into three types based on the extent of the lesion and patient needs: conservative surgery removes the endometriosis while preserving fertility, but this has a high recurrence rate; semi-radical surgery, which preserves ovarian function while removing the uterus, reduces the risk of recurrence but permanently impairs fertility; and radical surgery, which completely removes the lesion by removing the uterus and bilateral adnexa, but results in loss of ovarian function, menopausal symptoms, and long-term metabolic effects, severely impacting the patient's quality of life. Drug treatment options mainly include the following categories: non-steroidal anti-inflammatory drugs relieve pain by inhibiting prostaglandin synthesis, but cannot prevent disease progression, and long-term use may cause gastrointestinal and liver and kidney adverse reactions; oral contraceptives reduce estrogen levels by inhibiting ovulation, thereby inhibiting endometrial growth, but there is a risk of thrombosis and metabolic abnormalities; progestogens induce decidualization and atrophy of ectopic endometrium, but can easily lead to abnormal uterine bleeding and abnormal liver function; gonadotropin-releasing hormone analogues (GnRHa) significantly reduce estrogen levels by inhibiting the hypothalamic-pituitary-ovarian axis. Although the therapeutic effect is significant, the accompanying low estrogen state can cause vasomotor symptoms and bone loss, and reverse addition therapy is required to alleviate side effects.

[0005] β-Hydroxybutyrate (BHB), an endogenous ketone body, plays a crucial role in multiple physiological processes, including energy metabolism and inflammation regulation. BHB can be supplemented through injection or oral administration, or it can be converted through internal energy metabolism. Regarding energy metabolism, when the human body is in states of starvation, a low-carbohydrate diet, or intense exercise, fat breakdown accelerates, producing large amounts of fatty acids. These fatty acids undergo a series of metabolic processes in the liver to be converted into ketone bodies, of which BHB is a major component. Currently, research on endometriosis (endometriosis) focuses on traditional pathogenesis and clinical treatment strategies, while research on metabolic factors in this disease is limited. The relationship between BHB and endometriosis remains unclear. Although the regulatory role of metabolic abnormalities in gynecological diseases has gained increasing attention in recent years, the mechanisms linking BHB to endometriosis remain largely unresolved. Therefore, in-depth research on the relationship between BHB and endometriosis and exploring the feasibility of BHB as a potential treatment for endometriosis is crucial for enriching the theoretical understanding of endometriosis pathogenesis and providing new targets and drugs for clinical treatment. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention aims to provide the use of β-hydroxybutyric acid in the preparation of a drug for treating endometriosis.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: the present invention provides the use of β-hydroxybutyric acid in the preparation of a drug for treating endometriosis.

[0008] In some embodiments, the drug can significantly reduce endometriosis lesions during D4-D18 and D4-D28 after modeling surgery.

[0009] In some embodiments, the drug can increase the BHB content in the serum of patients with endometriosis to a level close to normal.

[0010] In some embodiments, BHB can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions. BHB can be used as the sole active ingredient or in combination with other drugs. Combination therapy can be achieved by administering the individual therapeutic components simultaneously, separately, or sequentially.

[0011] In some embodiments, β-hydroxybutyrate is the only active ingredient in the drug; the β-hydroxybutyrate can be used alone or in the form of a pharmaceutical composition.

[0012] In some embodiments, the pharmaceutical composition includes β-hydroxybutyrate and other pharmaceutically acceptable carriers.

[0013] The drug or pharmaceutical composition of the present invention can be administered by any general route as long as it can reach the target tissue. In other embodiments, the drug or pharmaceutical composition is formulated into a dosage form suitable for delivery to the target tissue via intraperitoneal or oral administration.

[0014] The medicament or pharmaceutical composition of the present invention can be conveniently presented in unit dosage form. The medicament or pharmaceutical composition of the present invention can be formulated into any suitable dosage form, such as, but not limited to, a preparation, tablet, capsule, gel, suppository, etc. The medicament or pharmaceutical composition of the present invention can also be formulated into a suspension in an aqueous, non-aqueous, or mixed medium.

[0015] In some embodiments, the medicine or pharmaceutical composition of the present invention is formulated into an injection dosage form or an oral dosage form; the oral dosage form is a powder, tablet, capsule, granule or oral solution.

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides the use of β-hydroxybutyrate in the preparation of a drug for treating endometriosis. In animal experiments, injection of BHB between days 4 and 18 after modeling significantly reduced endometriosis lesions. Clinical studies have found decreased serum BHB levels in endometriosis patients, suggesting that BHB supplementation may improve endometriosis symptoms, demonstrating its beneficial therapeutic effect on endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 The migration diagram and relative migration area diagram of normal endometrial stromal cells were detected at different time points after treatment with different concentrations of BHB; (a) is the migration diagram of normal endometrial stromal cells, and (b) is the relative migration area diagram of normal endometrial stromal cells.

[0020] Figure 2 The migration diagram and relative migration area diagram of ectopic endometrial stromal cells were detected at different time points after treatment with different concentrations of BHB; among them, (a) is the migration diagram of ectopic endometrial stromal cells, and (b) is the relative migration area diagram of ectopic endometrial stromal cells.

[0021] Figure 3 The figures are a comparison of the sizes of endometriosis lesions between the BHB treatment group and the control group during the period D4-D18 after modeling. (a) is a physical comparison between the BHB treatment group and the control group, (b) is a comparison of lesion weights, and (c) is a comparison of lesion volumes.

[0022] Figure 4 Comparison of the sizes of endometriosis lesions between the BHB treatment group and the control group during the period D4-D28 after modeling; (a) is the physical comparison between the BHB treatment group and the control group, (b) is the lesion weight comparison, and (c) is the lesion volume comparison.

[0023] Figure 5 This is a chart showing the detection of BHB levels in the serum of endometriosis patients and healthy people. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] The term "treat" generally refers to eliminating the disease, arresting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival of subjects suffering from the disease.

[0026] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any human or non-human animal, or cells thereof, suitable for use with the methods described herein, preferably a human or non-human mammal. In specific embodiments, the non-human mammal includes, for example, mice, rats, rabbits, pigs, and the like. In specific embodiments, the subject is human. In specific embodiments, the subject is susceptible to, suspected of having, or already has endometriosis.

[0027] The term "administer" may refer to providing a predetermined substance to a subject by any appropriate method. The term "therapeutically effective amount" may refer to an amount of an active ingredient or pharmaceutical composition that induces an animal or human to show a biological or medical response that is considered by a researcher, veterinarian, doctor or other clinician, and this amount may include the amount of an active ingredient or pharmaceutical composition that is used to induce the alleviation of a disease or condition to be treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present application may vary according to the desired effect. The amount of administration or intake may be administered in a variety of dosages and methods, by distributing compositions according to the subject's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of the disease, for example, once a day or multiple times a day.

[0028] The present invention will be further described below with reference to the embodiments.

[0029] Various cell lines, drugs and experimental animals used in the examples:

[0030] Cell line: Ectopic endometrial cells were obtained from surgically removed ectopic endometrial tissue and cultured in primary culture.

[0031] Drugs: β-Hydroxybutyric acid (BHB) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Sigma-Aldrich), catalog number 150-83-4.

[0032] Animals: Female 6-8 week old C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0033] Example 1: Effect of BHB on ectopic endometrial cell migration

[0034] 1. Experimental Procedure

[0035] Ectopic endometrial stromal cells were cultured at 5×10 5Cells were seeded into a 6-well plate, and DMEM F12 medium containing 10% fetal bovine serum was added. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. After the cells adhered to the wall, the tip of the pipette was used to scratch the cell layer along the line drawn on the back of the well plate. After the scratching was completed, the cells were washed three times with sterile PBS to remove the non-adherent cells, and then fresh culture medium was replaced. The horizontal scratch marks on the back of the 6-well plate were wiped off and photographed under a microscope. The samples were then divided into a control group and a BHB treatment group. The BHB treatment group was treated with BHB solutions with final concentrations of 2 mM, 5 mM, and 10 mM, respectively, and the control group was treated with an equal volume of culture medium. Pictures were taken after 12 h, 24 h, and 36 h of culture, and the results were analyzed.

[0036] 2. Experimental Results

[0037] To evaluate the effect of BHB on the migration ability of endometrial stromal cells, we performed in vitro migration experiments on normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs). Figure 1 and Figure 2 As shown, Figure 1 (a) is the migration diagram of normal endometrial stromal cells, and (b) is the relative migration area diagram of normal endometrial stromal cells; Figure 2 (a) is the migration diagram of ectopic endometrial stromal cells, and (b) is the relative migration area diagram of ectopic endometrial stromal cells. Compared with the control group, BHB treatment significantly inhibited the migration ability of ectopic endometrial stromal cells in a dose-dependent manner: as the BHB concentration gradually increased, the migration ability of eESCs continued to decline ( P <0.005), showing that BHB has a significant inhibitory effect on the migration of ectopic cells. In contrast, the migration ability of normal endometrial stromal cells did not change significantly under the same BHB treatment, that is, BHB had no significant effect on the migration of normal cells within the experimental concentration range. This result shows that BHB can selectively inhibit the migration of endometrial stromal cells under pathological conditions without affecting the migration behavior of normal cells, suggesting that BHB is specific and safe in the treatment of endometriosis. ns indicates no statistical difference, and "*" indicates P <0.05, “****” indicates P <0.0001.

[0038] Example 2: Effect of BHB on Lesion Size in Endometriosis Animal Model

[0039] 1. Experimental Procedure

[0040] 6-8 week old female C57BL / 6J mice were selected and after 1 week of adaptive feeding, endometriosis modeling surgery was performed. After the mice were anesthetized, the abdominal cavity was opened and part of the uterine tissue was cut into approximately 1 mm 3 The mice were randomly divided into a control group and a BHB-treated group (200 mg / kg intraperitoneal injection of BHB). Treatments were performed on days 4-18 and 4-28 after model establishment. The control group received an equal volume of PBS. After the respective treatment periods, the mice were anesthetized, the peritoneal cavity was opened, and the size of the ectopic lesions was observed and measured.

[0041] 2. Experimental Results

[0042] In an established endometriosis mouse model, we evaluated the effect of BHB intervention on the growth of ectopic lesions. The results were as follows: Figure 3 and Figure 4 shown. Figure 3 The figure is a comparison of the endometriosis lesion size between the BHB treatment group and the control group during the period of D4-D18 after modeling. Figure 3 (a) is a physical comparison diagram of the BHB treatment group and the control group, (b) is a comparison diagram of the lesion weight, and (c) is a comparison diagram of the lesion volume. Figure 4 This is a comparison of the endometriosis lesion size between the BHB treatment group and the control group during the period of D4-D28 after modeling; Figure 4 (a) shows a comparison of the BHB-treated and control groups, (b) shows a comparison of lesion weight, and (c) shows a comparison of lesion volume. Compared with the control group, BHB treatment significantly inhibited the growth of ectopic lesions. The size, weight, and volume of ectopic lesions in the BHB-treated group decreased significantly, with statistical significance ( P <0.05), indicating that BHB has a significant inhibitory effect on ectopic lesions. We further compared two different intervention time schemes: D4-D18 group and D4-D28 group. The results showed that although both groups could effectively inhibit lesion growth, the D4-D28 group (i.e., extended administration time) had a more significant therapeutic effect on all indicators, suggesting that the inhibitory effect of BHB is time-dependent: the longer the administration time, the stronger the anti-lesion growth effect. "*" indicates P <0.05, “**” indicates P <0.01.

[0043] Example 3: Comparison of serum BHB levels between endometriosis patients and normal subjects

[0044] 1. Experimental Procedure

[0045] Sixteen patients with clinically confirmed endometriosis served as the case group, and eight age- and weight-matched healthy women served as the control group. All participating patients provided written informed consent. Five milliliters of fasting venous blood was collected from both groups, and serum was separated by centrifugation. Serum BHB levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (purchased from Jiangsu Sumeike Biotechnology Co., Ltd., MK0532HA).

[0046] 2. Experimental Results

[0047] The BHB levels in the serum of endometriosis patients and normal healthy female controls were detected and compared to evaluate its metabolic changes in endometriosis. Figure 5 shown. Figure 5 The results show that the serum BHB level in endometriosis patients was significantly lower than that in the normal control group, which was 127.2 ± 7.8 μmol / L and 117.3 ± 5.3 μmol / L respectively, and the difference was statistically significant ( P = 0.0022). The above results suggest that the production or utilization of BHB may be inhibited in patients with endometriosis, which not only reflects the disorder of the ketone body metabolic pathway in their body, but also suggests that it may serve as a potential marker for endometriosis-related metabolic disorders. P <0.01.

[0048] In summary, the present invention demonstrates the following benefits: BHB, as a potential treatment for endometriosis, effectively reduces the size of endometriotic lesions in animal studies by inhibiting the migration of ectopic endometrial stromal cells and regulating inflammatory cytokine levels. Furthermore, clinical studies have shown decreased serum BHB levels in endometriosis patients, suggesting that BHB supplementation may improve their condition and has promising clinical application prospects. BHB occurs naturally in the body and is relatively safe, providing new insights and approaches for the treatment of endometriosis.

[0049] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of β-hydroxybutyric acid in the preparation of a medicament for treating endometriosis.

2. The use according to claim 1, characterized in that The drug can significantly reduce the endometriosis lesions during the period of D4-D18 and D4-D28 after modeling surgery.

3. The use according to claim 1, characterized in that The drug can increase the beta-hydroxybutyric acid content in the serum of patients with endometriosis.

4. The use according to any one of claims 1 to 3, characterized in that Beta-hydroxybutyrate is the only active ingredient in the drug.

5. The use according to any one of claims 1 to 3, characterized in that The β-hydroxybutyric acid is used alone or in the form of a pharmaceutical composition.

6. The use according to claim 5, characterized in that The pharmaceutical composition comprises beta-hydroxybutyric acid and other pharmaceutically acceptable carriers.

7. The use according to claim 5, characterized in that The drug or pharmaceutical composition is formulated into a dosage form suitable for delivery to the target tissue via intraperitoneal or oral administration.

8. The use according to claim 7, characterized in that The medicine or pharmaceutical composition is formulated as an injection form.

9. The use according to claim 7, characterized in that The drug or pharmaceutical composition is formulated as an oral dosage form.

10. The use according to claim 9, characterized in that The oral dosage form is powder, tablet, capsule, granule or oral liquid.

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