Application of targeted proline metabolic enzymes in the treatment of endometriosis

Abstract

This disclosure belongs to the field of pharmaceutical technology, and specifically relates to the application of targeted proline metabolic enzymes in the treatment of endometriosis. This disclosure, through in vitro experiments and EMS animal models, verifies that the proline content in ectopic endometrial stromal cells increases, the expression of proline synthesis metabolic enzymes is elevated, and fibrosis is significantly activated. This disclosure creatively repurposes proline metabolic enzyme inhibitors for EMS treatment. Proline metabolic enzyme inhibitors specifically inhibit the enzyme activity of key proline metabolism enzymes, blocking the synthesis pathway from glutamate to proline, and directly reducing intracellular proline content. Since proline is a major component amino acid of collagen, the inhibition of proline synthesis directly inhibits collagen synthesis and the expression of fibrosis markers in endometrial stromal cells, ultimately achieving the therapeutic effect of inhibiting fibrosis in EMS ectopic lesions and reducing ectopic lesion growth.

Description

Technical Field

[0001] This disclosure belongs to the field of pharmaceutical technology, and specifically relates to the application of targeted proline metabolic enzymes in the treatment of endometriosis. Background Technology

[0002] Endometriosis (EMS) is a common gynecological disease characterized by the presence of endometrial-like lesions outside the uterine cavity. This manifests as ectopic growth of endometrial tissue in locations such as the ovaries, pelvic peritoneum, and intestines, exhibiting cyclical bleeding, proliferation, and fibrosis in response to hormonal fluctuations during the menstrual cycle. Fibrosis is a core pathological feature of EMS, leading to adhesions, pain, and infertility. It significantly increases the difficulty and risk of surgery, increasing the potential for ovarian damage and impacting postoperative fertility. Multiple studies have indicated that endometriosis is a potential risk factor for various diseases, including cancer, asthma, autoimmune diseases, and cardiovascular diseases.

[0003] Diagnosing endometriosis remains a clinical challenge. Diagnosis typically relies on clinical symptoms and signs, imaging studies, laparoscopy, and serum CA125 levels. The gold standard is generally considered to be laparoscopic observation of the lesion's morphology, combined with histopathological examination (the lesion may show endometrial glands and stroma, accompanied by inflammation and fibrosis). However, in some clinical cases, histopathological evidence is lacking, and there are currently no clear molecular markers to aid in histopathological detection.

[0004] Treatment for endometriosis mainly falls into two categories: surgical and pharmacological. Surgical treatment is often ineffective, with a recurrence rate as high as 75% within 5 years. Drug therapy aims to inhibit lesion progression. Common drugs are primarily classified into five classes: nonsteroidal anti-inflammatory drugs (NSAIDs), oral contraceptives, high-potency progestins, androgen derivatives, and gonadotropin-releasing hormone agonists. Because drug therapy mainly controls the hypothalamic-pituitary-ovarian reproductive endocrine axis, it has strong side effects and is not suitable for young patients with fertility needs. More regrettably, there are currently no ideal molecular targets for targeted therapy of endometriosis. Therefore, the discovery and functional identification of important molecular targets are crucial for breaking this deadlock and will lay the molecular theoretical foundation for subsequent drug development.

[0005] Fibrosis in ectopic lesions is primarily driven by excessive deposition and abnormal remodeling of collagen in the extracellular matrix. During this pathological process, collagen metabolism in ectopic endometrial stromal cells is activated, and their ability to synthesize collagen is continuously enhanced. Activated ectopic endometrial stromal cells secrete large amounts of procollagen into the extracellular space, where it undergoes cross-linking and maturation under the action of specific enzymes, forming a stable and difficult-to-degrade dense fibrous network, ultimately leading to fibrous tissue proliferation, adhesion formation, and progressive tissue sclerosis. Proline is a major component amino acid of collagen, and its key metabolic enzymes, P5CS and PYCR1, can catalyze the synthesis of pyrroline-5-carboxylate (Δ¹-Pyrroline-5-carboxylate, P5C) from glutamate, further generating proline. Existing studies have shown that collagen content is significantly elevated and fibrosis is pronounced in ectopic lesions of endometrial spondylosis (EMS), but there is a lack of research on the relationship between proline metabolism and collagen synthesis and fibrosis, and a strategy targeting fibrosis to treat EMS is also lacking. Summary of the Invention

[0006] The purpose of this disclosure is to provide the application of targeting proline metabolic enzymes in the treatment of endometriosis. This invention, through in vitro experiments and EMS animal models, validates that ectopic endometrial stromal cells (eESCs) exhibit increased proline content, elevated expression of proline biosynthetic enzymes, and significant activation of fibrosis. The inhibitors of key proline biosynthetic enzymes, YG1702 and PYCR1-IN-1, can inhibit collagen synthesis in endometrial stromal cells (ESCs), reduce the expression of fibrosis markers, and decrease the weight of ectopic lesions in animal models. These results suggest that these inhibitors can be further used to prepare drugs for the treatment of endometriosis.

[0007] The objective of this disclosure is achieved through the following technical solution: In a first aspect, this disclosure provides the use of targeting proline-metabolizing enzymes in the treatment of endometriosis, said use comprising at least one of the following: (1) Application in the preparation of products for endometriosis by targeting proline metabolic enzymes; (2) Use in the preparation of products for relieving and / or treating endometriosis; (3) Application in products that serve as metabolic targets for the treatment of endometriosis.

[0008] In some specific embodiments of this disclosure, the targeted proline metabolic enzyme refers to a proline metabolic enzyme inhibitor that reduces collagen deposition and ectopic lesion growth in endometriosis by inhibiting proline metabolism, collagen synthesis, and fibrosis.

[0009] In some embodiments of this disclosure, the proline metabolic enzyme is pyrrololine-5-carboxylic acid synthase and / or pyrrololine-5-carboxylic acid reductase 1.

[0010] In some specific embodiments of this disclosure, the proline metabolic enzyme inhibitor is a specific small molecule inhibitor, and further, the specific small molecule inhibitor is YG1702 and / or PYCR1-IN-1.

[0011] In some specific embodiments of this disclosure, the product includes one or more of the following: drugs, reagents, kits, chips, test strips, membrane strips, and detection platforms.

[0012] In a second aspect of this disclosure, the use of proline metabolic enzyme inhibitors in the preparation of medicaments for relieving and / or treating endometriosis is provided.

[0013] In some specific embodiments of this disclosure, the application includes administering an effective amount of a proline-metabolizing enzyme inhibitor to a subject in need, and further, administering an effective amount of YG1702 and / or PYCR1-IN-1.

[0014] In a third aspect of this disclosure, this disclosure provides a medicament for alleviating and / or treating endometriosis, said medicament comprising a proline metabolizing enzyme inhibitor, further wherein said proline metabolizing enzyme inhibitor is YG1702 and / or PYCR1-IN-1.

[0015] In some specific embodiments of this disclosure, the medicament further includes a pharmaceutically acceptable carrier or excipient, and further, the medicament is used alone or in combination with other medicaments.

[0016] In a fourth aspect of this disclosure, a pharmaceutical composition for relieving and / or treating endometriosis is provided, the pharmaceutical composition comprising YG1702 and / or PYCR1-IN-1, further comprising a pharmaceutically acceptable carrier or excipient, and further, the pharmaceutical composition being used alone or in combination with other pharmaceuticals.

[0017] The technical solution provided in this disclosure has the following technical contributions: (1) Based on a novel logical chain of “inhibitors targeting proline metabolic enzymes regulate proline synthesis → proline is the main component of collagen → inhibiting the proline metabolic axis can reduce collagen deposition → treating EMS fibrosis”, this disclosure first discovered and verified through in vitro experiments and EMS animal models that key proline metabolic enzymes (such as P5CS and PYCR1) are novel metabolic targets for EMS treatment, providing a new direction for EMS treatment.

[0018] (2) This disclosure creatively reuses proline metabolic enzyme inhibitors for EMS treatment. Proline metabolic enzyme inhibitors block the synthesis pathway from glutamate to proline by specifically inhibiting the enzyme activity of key proline metabolism enzymes (such as P5CS and PYCR1), thereby directly reducing the intracellular proline content. Since proline is a major component amino acid of collagen (accounting for 20% to 30% of collagen amino acid residues), the inhibition of proline synthesis directly inhibits collagen synthesis and the expression of fibrosis markers (COL1A1, α-SMA) in endometrial stromal cells, ultimately achieving the therapeutic effect of inhibiting fibrosis of EMS ectopic lesions and reducing the growth of ectopic lesions.

[0019] (3) This disclosure proposes a novel strategy for treating EMS by targeting the proline metabolic axis, which is different from existing traditional treatments such as hormone therapy and surgery, and is expected to achieve precise treatment with low side effects. Attached Figure Description

[0020] Figure 1 The values ​​represent the relative enrichment levels of proline and glutamate in normal and ectopic endometrium. ESC: normal endometrial stromal cells; eESC: ectopic endometrial stromal cells; Relative levels: relative expression abundance; ***: P < 0.001; Glutamate; Proline.

[0021] Figure 2 This study describes the expression of pyrrololine-5-carboxylic acid synthase (P5CS) and pyrrololine-5-carboxylic acid reductase 1 (PYCR1) in normal and ectopic endometrium. In the figures, Ctrl represents the normal endometrium group; EMs represents the ectopic endometrium group; Isotype represents the blank control; 10X, 20X, and 40X are objective magnifications, and the legend scale bars are 100μm, 50μm, and 20μm, respectively. The average positive rates of PYCR1 and P5CS in the normal endometrium group were 3.20% and 4.30%, respectively, while the average positive rates in the ectopic endometrium group were 17.8% and 17.1%, respectively.

[0022] Figure 3 These are the results of Masson staining of normal endometrium and ectopic endometrium. In the diagram, Ctrl represents the normal endometrium group; EMs represents the ectopic endometrium group; 4X, 10X, and 20X are the objective lens magnifications; blue represents collagen fibers, red represents muscle fibers, and blue-black represents cell nuclei. The average collagen area percentage in the normal endometrium group was 21.5%, while the average collagen area percentage in the ectopic endometrium group was 35.0%.

[0023] Figure 4YG1702 and PYCR1-IN-1 inhibit the expression of collagen and the fibrosis marker α-smooth muscle actin (α-SMA) in endometrial stromal cells (ESCs). Ctrl: control group; YG1702: YG1702 treatment group; PYCR1-IN-1: PYCR1-IN-1 treatment group; COL1A1: COL1A1 (type I collagen α1 chain); α-SMA: α-smooth muscle actin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Relative intensity: relative expression level (with GAPDH as the reference standard); ***: P < 0.001.

[0024] Figure 5 YG1702 and PYCR1-IN-1 significantly inhibited the weight of ectopic lesions and suppressed collagen synthesis and fibrosis in ectopic lesions. Figure 5 Image A shows an ectopic lesion. Figure 5 B represents the weight statistics of ectopic lesions (ectopic lesions cannot be palpated or detected on the body surface during their growth period, and can only be measured once after removal; their shape is irregular, so only the weight is measured). Figure 5 C represents the peritoneal cavity of the model mice. Figure 5 D represents Masson staining after preparing a slide of the ectopic lesion. Figure 5 E represents the statistical results of Masson staining. Figure 5 F represents the immunohistochemical results of ectopic lesion slides, detecting COL1A1 and α-SMA. Wherein, Ctrl: control group EMS mice; YG1702: YG1702 intraperitoneal injection group EMS mice, 45 mg / kg, once every 3 days; PYCR1-IN-1: PYCR1-IN-1 intraperitoneal injection group EMS mice, 10 mg / kg, once every 3 days; ***: P < 0.001, ****: P < 0.0001; Isotype: blank control; 10X, 20X, and 40X are objective magnifications, with legend scale bars of 100 μm, 50 μm, and 20 μm, respectively. Detailed Implementation

[0025] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0026] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology.

[0027] In the following embodiments, unless otherwise specified, the terms used are explained as follows: In this disclosure, unless otherwise specified, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0028] In this disclosure, unless otherwise specified, the term "ectopic endometrial stromal cells (eESC)" refers to a population of endometrial stromal cells (typical markers CD10 / MME, positive for Vimentin, negative for CK) that should be located in the uterine cavity, but which have ectopically colonized external sites such as the ovary, peritoneum, intestine, and bladder, forming lesions; they often coexist with ectopic endometrial epithelial cells, constituting the main supporting structure of ectopic lesions.

[0029] In this disclosure, unless otherwise specified, the term "P5CS" refers to pyrroline-5-carboxylate synthase, encoded by gene ALDH18A1, located on human chromosome 10q24.31. The expressed P5CS protein is a multifunctional enzyme, possessing both γ-glutamyl kinase and glutamate-5-semialdehyde dehydrogenase activities. It is the only rate-limiting enzyme in proline synthesis, and its expression and activity directly determine the rate of proline synthesis. Starting with L-glutamate as a substrate, the two-step sequential catalytic synthesis of pyrroline-5-carboxylic acid (P5C) is the core upstream step in the de novo synthesis of proline. Without the catalytic action of P5CS, endogenous proline synthesis would be completely inhibited.

[0030] In this disclosure, unless otherwise specified, the term "YG1702" refers to a specific small molecule inhibitor of pyrroline-5-carboxylic acid synthase (P5CS / ALDH18A1). YG1702 directly inhibits the enzyme activity of P5CS by binding to its catalytic domain, functionally blocking the conversion of glutamate to P5C and inhibiting proline synthesis at its source.

[0031] In this disclosure, unless otherwise specified, the term "PYCR1" refers to a downstream key enzyme in the proline biosynthesis pathway, forming a continuous catalytic pathway with the upstream P5CS. PYCR1 catalyzes the conversion of pyrroline-5-carboxylic acid (P5C) to proline, which is the final key reaction in the synthesis of proline from glutamate, providing the core raw material for collagen synthesis.

[0032] In this disclosure, unless otherwise specified, the term "PYCR1-IN-1" is a specific small molecule inhibitor of pyrroline-5-carboxylic acid reductase 1 (PYCR1), forming a dual-targeting inhibitor of the proline biosynthesis pathway upstream and downstream with YG1702. PYCR1-IN-1 directly reduces the proline content in endometrial stromal cells by specifically inhibiting the enzymatic activity of PYCR1, blocking the conversion of P5C to proline.

[0033] In this disclosure, unless otherwise specified, the terms "pharmaceutical composition" or "composition" may refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components.

[0034] In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components comprising the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0035] In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients, except for those incompatible with the proline metabolism inhibitors of this disclosure, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this disclosure.

[0036] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0037] Example 1: In vitro experiments confirmed that glutamate and proline were significantly enriched in ectopic endometrial stromal cells. Normal endometrial tissue (n=12) and ectopic lesions from EMS patients (n=9) were collected.

[0038] Inclusion and exclusion criteria: Inclusion criteria for the normal endometrial group: A. Patients' age group matched with those of patients with endometrial fibroids; B. Patients with simple uterine fibroids confirmed by our hospital's pathology or patients who underwent diagnostic curettage without endometrial lesions.

[0039] Exclusion criteria for the normal endometrial group: A. The patient has received hormone therapy within the past 6 months; B. The patient has other malignant lesions, chronic diseases, or immune diseases; C. The patient has not signed the "Informed Consent Form".

[0040] Inclusion criteria for ectopic lesions in the EMs group: A. Patients with EMs who were pathologically confirmed as endometrioid cysts in our hospital; B. Patients who had not received hormone therapy within the past 6 months.

[0041] Exclusion criteria for ectopic lesions in the EMs group: A. The patient has received hormone therapy within the past 6 months; B. The patient has other malignant lesions, chronic diseases, or immune diseases; C. The patient has not signed the "Informed Consent Form".

[0042] ESCs were isolated and cultured in vitro (isolation and identification methods were the same as in previous literature by our team, J Mol Endocrinol, 2011; Fertil Steril, 2012). After two passages, cells were collected after reaching confluence. Cells were lysed with ddH2O, extracted with methanol, and centrifuged at 20,000g for 10 minutes. The supernatant was collected and evaporated to dryness at no higher than 45℃ using a rotary evaporator. The metabolites were collected and analyzed by liquid chromatography-tandem mass spectrometry (the instrumentation platform in this experiment was LC-MS (Thermo, Ultimate3000LC, Orbitrap Elite), and the separation column was C2000C4). 18 The chromatographic column was Hypergod C18 (100x4.6mm 3um). Chromatographic separation conditions were: column temperature 40 °C; flow rate 0.3 mL / min; mobile phase composition A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid. The peak intensity of various metabolites was analyzed using Analyst software. Experimental results showed that the concentrations of glutamate and proline in ectopic endometrial stromal cells (eESCs) were significantly higher than those in normal endometrial stromal cells (ESCs), and the differences were statistically significant. Figure 1 ).

[0043] Example 2: In endometriosis ectopic lesions, P5CS and PYCR1 levels were significantly elevated compared to normal endometrium. Normal endometrial tissue and ectopic lesions from endometriosis patients were collected. After washing with PBS, the tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunohistochemical staining was performed (immunohistochemical antibodies: P5CS purchased from Abcam, catalog number ab315816, 1:1000; PYCR1 purchased from CST, catalog number 37635T, 1:1000, incubated overnight at 4°C; secondary antibody Anti-rabbit IgG purchased from CST, catalog number 7074S, 1:3000, incubated at room temperature for 1 hour). The results confirmed that P5CS and PYCR1 expression was low in normal endometrial tissue (Ctrl), while P5CS and PYCR1 were mainly expressed in stromal cells in ectopic lesions (EMs) from endometriosis patients. Compared with normal endometrial tissue (Ctrl), P5CS and PYCR1 expression were significantly increased in ectopic lesions (EMs). Figure 2 ).

[0044] Example 3: In endometriosis, collagen content was significantly increased compared to normal endometrium. Normal endometrial tissue and ectopic lesions from EMS patients were collected, washed with PBS, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Masson staining (Beijing Solarbio Science & Technology Co., Ltd., catalog number G1346) showed that normal endometrial tissue (Ctrl) had a low collagen content (blue area), while ectopic lesions (EMs) had a higher proportion of collagen area, a more obvious collagen structure, and significantly increased collagen deposition in the extracellular matrix. Figure 3 ).

[0045] Example 4: YG1702 and PYCR1-IN-1 inhibit collagen synthesis in ESCs Endometrial stromal cells (ESCs) were cultured in vitro. The control group (Ctrl) was treated with the control solvent, while the experimental groups (YG1702 and PYCR1-IN-1) were treated with YG1702 (10 μmol / L, purchased from MedChemExpress) and PYCR1-IN-1 (10 μmol / L, purchased from MedChemExpress), respectively, for 48 hours. Cells were collected, total protein was extracted, and Western blotting was performed. The results showed that both YG1702 and PYCR1-IN-1 significantly reduced the expression of COL1A1 and α-SMA in ESCs, confirming that YG1702 and PYCR1-IN-1 inhibited type I collagen synthesis and fibrosis in ESCs. Figure 4 ).

[0046] Example 5: Animal experiments verified that YG1702 and PYCR1-IN-1 inhibited the weight of ectopic lesions and reduced collagen content and fibrosis in ectopic lesions. An EMS mouse model was constructed using healthy 7-week-old female C57B / L6 mice (Shanghai Jiesijie Laboratory Animal Co., Ltd.). Recipient mice (n=18) and donor mice (n=12) were induced twice preoperatively with intramuscular injections of estrogen (estradiol benzoate 3 μg / 20g·time, PBS solvent) (day 1 and day 4). The uterus of the donor mice was removed, minced to <1mm, and injected intraperitoneally into the recipient mice (uterine fragments from one donor mouse were injected into two recipient mice). The recipient mice were divided into three groups, and every 3 days, they were injected intraperitoneally with PBS or YG1702, or PYCR1-IN-1, respectively. After 14 days of postoperative observation, mice were euthanized by cervical dislocation, and the peritoneal cavity was opened to examine the formation and weight of ectopic lesions. The ectopic lesions were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to immunohistochemistry and Masson staining. The experimental results showed that, compared with the control group, the weight of ectopic lesions in mice treated with YG1702 and PYCR1-IN-1 was significantly reduced (e.g., Figure 5 A, 5B), and the collagen content, COL1A1 expression, and α-SMA expression in ectopic lesions were all decreased ( Figure 5 D, Figure 5 F).

[0047] The above specific embodiments are merely illustrative of the contents of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. The application of targeted proline-metabolizing enzymes in the treatment of endometriosis, characterized in that, The application includes at least one of the following: (1) Application in the preparation of products for endometriosis by targeting proline metabolic enzymes; (2) Use in the preparation of products for relieving and / or treating endometriosis; (3) Application in products that serve as metabolic targets for the treatment of endometriosis.

2. The application according to claim 1, characterized in that, The targeted proline metabolic enzyme refers to proline metabolic enzyme inhibitors, which reduce collagen deposition and ectopic lesion growth in endometriosis by inhibiting proline metabolism, collagen synthesis, and fibrosis.

3. The application according to claim 1 or 2, characterized in that, The proline metabolic enzyme is pyrrolinoline-5-carboxylic acid synthase and / or pyrrolinoline-5-carboxylic acid reductase 1.

4. The application according to claim 2, characterized in that, The proline metabolic enzyme inhibitor is a specific small molecule inhibitor, and further, the specific small molecule inhibitor is YG1702 and / or PYCR1-IN-1.

5. The application according to claim 1, characterized in that, The products include one or more of the following: drugs, reagents, reagent kits, chips, test strips, membrane strips, and detection platforms.

6. Use of proline metabolic enzyme inhibitors in the preparation of drugs for relieving and / or treating endometriosis.

7. The application according to claim 6, characterized in that, The application includes administering an effective amount of a proline-metabolizing enzyme inhibitor to a subject in need, and further, administering an effective amount of YG1702 and / or PYCR1-IN-1.

8. A medicine for relieving and / or treating endometriosis, characterized in that, The drug includes a proline metabolic enzyme inhibitor, and more specifically, the proline metabolic enzyme inhibitor is YG1702 and / or PYCR1-IN-1.

9. The medicament according to claim 8, characterized in that, The drug may also include pharmaceutically acceptable carriers or excipients, and further, the drug may be used alone or in combination with other drugs.

10. A pharmaceutical composition for relieving and / or treating endometriosis, characterized in that, The pharmaceutical composition includes YG1702 and / or PYCR1-IN-1, and further, the pharmaceutical composition includes a pharmaceutically acceptable carrier or excipient, and further, the pharmaceutical composition is used alone or in combination with other pharmaceutical compositions.

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