Application of niclosamide in preparation of medicine for treating endometriosis

Abstract

The invention discloses application of niclosamide in preparation of a medicine for treating endometriosis, and belongs to the technical field of biological medicine. In-vivo and in-vitro experiments prove that the niclosamide can down-regulate the expression of primary endometriosis mesenchymal cells CD47 through a JAK / STAT3 pathway, enhance the phagocytosis of macrophages and play a role in effectively treating endometriosis; the invention provides the preparation method of the niclosamide microneedle, the preparation method is simple, the prepared LPHM-coated DDI NPs shows synergistic cytotoxicity and apoptosis induction activity in endometriosis mesenchymal cells, growth and recurrence of a focus of a subcutaneous endometriosis mouse model can be effectively inhibited through tumor ablation and anti-angiogenesis, and the niclosamide microneedle has a good application prospect. Rapid and effective treatment of endometriosis is realized in a microneedle patch mode.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of niclosamide in preparing a drug for treating endometriosis. Background Art

[0002] Abdominal wall endometriosis (AWE) is a special type of extrapelvic endometriosis, with glands and stroma located in various layers of the abdominal wall. AWE often occurs after cesarean section, and patients often present with abdominal wall pain during menstruation, but surgery is still the main treatment method so far. AWE often involves fascia and invasion of the rectus abdominis muscle requires patch repair to complete the surgery. The larger and deeper the nodules formed, the higher the recurrence rate. Even postoperative hormone therapy cannot effectively reduce the recurrence rate.

[0003] At present, the drugs used to treat AWE are mainly drugs that inhibit estrogen, including oral contraceptives, high-efficiency progestins and gonadotropin-releasing hormone agonists. Among them, gonadotropin-releasing hormone agonists have serious side effects of low estrogen and can only be used as a short-term treatment. During the treatment, patients need to endure problems such as amenorrhea and infertility. High-efficiency progestins cannot be used for a long time due to the risks of low estrogen, amenorrhea, irregular uterine bleeding, breast cancer and hypercoagulation. Oral contraceptives also have risks such as irregular uterine bleeding and breast cancer.

[0004] Researching and developing new drugs, including new uses of old drugs, to treat abdominal wall endometriosis, so that they have effective therapeutic effects while avoiding adverse effects such as affecting the patient's sex hormone levels, is a key issue in the current clinical treatment of abdominal wall endometriosis. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide the use of niclosamide in the preparation of a drug for treating abdominal wall endometriosis, so as to solve the problem that existing drugs for treating AWE have adverse effects such as affecting the patient's sex hormone levels during the treatment process, and lack of effective and safe drugs for treating AWE.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] Application of niclosamide in the preparation of medicines for treating endometriosis.

[0008] The beneficial effects of the present invention are as follows: the present invention has verified through in vivo and in vitro experiments that niclosamide can downregulate the expression of CD47 in primary endometriotic stromal cells through the JAK / STAT3 pathway, enhance the phagocytic effect of macrophages, and play an effective role in treating endometriosis.

[0009] A drug for treating endometriosis, the active ingredient of which is niclosamide.

[0010] Furthermore, the dosage form of the drug includes oral preparations or injection preparations.

[0011] A niclosamide microneedle patch, wherein the active ingredient comprises niclosamide.

[0012] The above-mentioned niclosamide microneedle patch is prepared by the following steps:

[0013] (1) Mixing mesoporous silica nanoparticles and leukocyte / platelet hybrid membranes in a solution, adding near-infrared fluorescent dye and niclosamide, mixing well and freeze-drying to prepare composite nanoparticles LPHM@DDI NPs;

[0014] (2) mixing the composite nanoparticles obtained in step (1) and the hyaluronic acid solution evenly, placing the mixture in a microneedle mold, and centrifuging the mixture;

[0015] (3) adding hyaluronic acid solution to the microneedle mold after centrifugation in step (2) for a second time, centrifuging again, and finally vacuum drying to obtain.

[0016] The beneficial effects of adopting the above-mentioned further technical scheme are as follows: the present invention establishes a bionic nano-platform based on leukocyte / platelet hybrid membrane (LPHM) and dendritic macroporous mesoporous silica nanoparticles (DLMSNs), and co-loads near-infrared fluorescent dye and niclosamide into the macropores of DLMSNs. Through the mediation of LPHM, LPHM@DDI NPs exhibit excellent targeting ability and very high photothermal therapy performance both in vitro and in vivo. Under near-infrared laser irradiation, LPHM@DDI NPs exhibit synergistic cytotoxicity and apoptosis-inducing activity in endometriotic stromal cells. The growth and recurrence of lesions in a subcutaneous endometriosis mouse model can be effectively inhibited through tumor ablation and anti-angiogenesis, and rapid and effective treatment of endometriosis can be achieved by microneedle patch.

[0017] Furthermore, the near-infrared fluorescent dye is IR780.

[0018] Furthermore, in step (1), the molar volume ratio of the composite nanoparticles to the hyaluronic acid solution is 1-5 μmol: 20-100 μL; and the mass concentration of the hyaluronic acid solution is 13-18%.

[0019] Furthermore, the centrifugation conditions in step (1) are: first centrifugation at 3-5°C, 1000-2000 rpm for 5-20 min, and then centrifugation at 3-5°C, 3000-5000 rpm for 5-20 min.

[0020] Furthermore, the volume ratio of the hyaluronic acid solution added for the second time in step (2) to the mixed solution in the microneedle mold in step (1) is 20-40:40-60; the mass concentration of the hyaluronic acid solution is 8-12%.

[0021] Furthermore, the conditions for the second centrifugation in step (2) are: centrifugation at 3-5°C and 2000-4000 rpm for 10-30 min.

[0022] Application of the above-mentioned niclosamide microneedle patch in the preparation of drugs or medical devices for treating endometriosis.

[0023] The present invention has the following beneficial effects:

[0024] (1) The present invention has verified through in vivo and in vitro experiments that niclosamide can downregulate the expression of CD47 in primary endometriotic stromal cells through the JAK / STAT3 pathway, enhance the phagocytic effect of macrophages, and effectively treat abdominal wall endometriosis.

[0025] (2) The present invention provides a method for preparing a niclosamide microneedle patch. The preparation method is simple. The prepared LPHM@DDI NPs exhibit synergistic cytotoxicity and apoptosis-inducing activity in endometriotic stromal cells, and can effectively inhibit the growth and recurrence of lesions in a subcutaneous endometriosis mouse model through tumor ablation and anti-angiogenesis, thereby achieving rapid and effective treatment of endometriosis by means of a microneedle patch. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The expression of CD47 in ectopic tissues of patients with endometriosis in Example 1, wherein A is the immunohistochemistry result, B is the qPCR result, C is the immunofluorescence result, and D is the immunofluorescence co-localization expression quantification diagram of CD47 and CD10 by ImgageJ statistics;

[0027] Figure 2 The expression of JAK STAT3 in endometriosis tissue in Example 1, wherein A is the immunofluorescence expression result of JAK STAT3, B is the quantification diagram of JAK STAT3 co-localization expression, and C is the expression result of macrophage marker CD68 and M2 macrophage marker CD206;

[0028] Figure 3The results of the effect of niclosamide on the function of endometriotic stromal cells in Example 2 are shown in Figure 2, wherein A is the result of optical microscopy Vimentin verification of stromal cells, B is the effect of niclosamide on the viability of endometriotic stromal cells, C is the effect of niclosamide on the activity of STAT3 in primary endometriotic stromal cells, and D is the effect of niclosamide on the invasiveness of primary endometriotic stromal cells;

[0029] Figure 4 The results of the effect of niclosamide on the lesions of the endometriosis mouse model in Example 2 are shown, wherein A is a lesion size diagram, B is a lesion mass diagram, C is a diagram showing changes in CD47 expression in the lesions, D is a diagram showing changes in JACK STAT3 expression in the lesions, and E is a diagram showing tissue immunofluorescence results. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] Example 1: Detection of the correlation between JAK / STAT3 / CD47 expression and endometriosis staging

[0032] 1. Experimental Methods

[0033] (1) Clinical case collection: Married patients who were pathologically diagnosed with endometriosis and required curettage during surgery at the General Hospital of Ningxia Medical University from January to August 2023 were collected. The control group consisted of patients with normal endometrium during hysteroscopy for infertility surgery. The age range was 26-45 years. The following conditions were excluded: inflammation, tuberculosis, borderline and malignant diseases, combined internal medicine diseases, and the use of hormones or related drugs in the past three months.

[0034] (2) Tissue specimens were collected from two groups of patients, including endometrium (CON) from the control group, and eutopic endometrium (EU) and ectopic endometrium (CU) from the endometriosis group. Immunohistochemistry, immunofluorescence, WB, and qPCR were used to detect the distribution of macrophages and the expression of JAK / STAT3 / CD47 and their correlation with the staging of endometriosis.

[0035] 2. Experimental Results

[0036] The experimental results are as follows Figure 1 and Figure 2 shown.

[0037] according to Figure 1As shown in Figure A, CD47 is significantly expressed in ectopic tissue and is significantly higher than that in eutopic endometrium and normal endometrium. According to the qPCR results ( Figure 1 Figure B) shows that CD47 expression is significantly increased in ectopic tissues; Figure 1 The immunofluorescence display results and expression quantification diagrams in Figures C and D show that the expression of CD47 is significantly increased in the ectopic tissues of patients with endometriosis.

[0038] according to Figure 2 As shown in Figures A and B, the expression of JAK STAT3 in ectopic tissues was significantly increased compared with that in eutopic endometrium and normal endometrium, and the expression of macrophage marker CD68 and M2 macrophage marker CD206 in ectopic tissues was decreased.

[0039] Example 2: The therapeutic effect of niclosamide on endometriosis

[0040] (1) Effects of niclosamide on the function of primary endometriotic stromal cells

[0041] After adding different concentrations of niclosamide to the primary endometrial stromal cells extracted in Example 1, cell proliferation was detected by CCK8, cell apoptosis was detected by flow cytometry, cell invasion and migration were detected by scratch and Transwell experiments, and differential expression of cytokines was detected by inflammatory factor chip. Overexpression or silencing of JACK / STAT in endometriotic stromal cells in different conditioned medium to detect CD47 expression verified that niclosamide regulates CD47 expression in endometriotic stromal cells through the JACK / STAT pathway to exert a therapeutic effect.

[0042] The experimental results are as follows Figure 3 shown.

[0043] Depend on Figure 3 It can be seen that by adding different concentrations of niclosamide, the viability of endometriotic stromal cells, the activity of STAT3 in primary endometriotic stromal cells and the invasiveness of primary endometriotic stromal cells can be effectively reduced, and with the increase of niclosamide concentration, the viability of endometriotic stromal cells, the activity of STAT3 and the invasiveness gradually decreased. The results show that niclosamide can effectively inhibit the growth of endometrial stromal cells.

[0044] (2) Niclosamide inhibits the growth of lesions in endometriosis mouse model

[0045] The EM mouse model was established by intraperitoneally injecting minced uterine fragments from a donor mouse evenly into the abdominal cavity of recipient mice.

[0046] The mice were divided into an untreated control group and an oral niclosamide group (200 mg / kg / day). The size, volume and weight of the lesions formed in the mice were detected after 14 days of culture, and the expression of JACK / STAT3 / CD47 in the lesions was detected by WB, immunofluorescence and immunohistochemistry.

[0047] The experimental results are as follows Figure 4 shown.

[0048] Depend on Figure 4 Figures A and B show that the size and volume of endometriosis lesions in mice treated with niclosamide were significantly reduced compared with those in the control group. Figure 4 As shown in Figures C-E, the expression of CD47 and JACK STAT3 in the lesions formed by mice orally taking niclosamide was significantly reduced. The results show that niclosamide can effectively inhibit the growth of endometriosis lesions by reducing the expression of CD47 and JACK STAT3, and play an effective role in the treatment of endometriosis.

[0049] Embodiment 3:

[0050] A method for preparing a niclosamide microneedle patch comprises the following steps:

[0051] (1) The mesoporous silica nanoparticles and the leukocyte / platelet hybrid membrane were mixed evenly in a solution, near-infrared fluorescent dye IR780 and niclosamide were added, the mixture was mixed evenly and then freeze-dried for 24 hours to obtain composite nanoparticles LPHM@DDI NPs;

[0052] (2) 2.5 μmol of the composite nanoparticles obtained in step (1) and a hyaluronic acid solution with a mass fraction of 15% were mixed evenly and placed in a microneedle mold, and centrifuged, first at 4°C and about 1500 rpm for 5 min each, and then at 4°C and about 4000 rpm for 5 min each;

[0053] (3) Add 30 μL of 10% hyaluronic acid solution to the microneedle mold after centrifugation in step (2), centrifuge again, and centrifuge at 4° C. and 3000 rpm for 10 min each, and finally vacuum dry overnight to obtain the microneedle mold.

[0054] The LPHM@DDI NPs prepared in the present invention exhibit synergistic cytotoxicity and apoptosis-inducing activity in endometriotic stromal cells, can effectively inhibit the growth and recurrence of lesions in a subcutaneous endometriosis mouse model through tumor ablation and anti-angiogenesis, and can achieve rapid and effective treatment of endometriosis through a microneedle patch.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of niclosamide in the preparation of drugs for the treatment of endometriosis.

2. A drug for treating endometriosis, characterized in that: The active ingredient includes niclosamide.

3. The drug for treating endometriosis according to claim 2, characterized in that: The dosage form of the drug includes oral preparations or injection preparations.

4. A niclosamide microneedle patch, characterized in that: The active ingredient includes niclosamide.

5. The method for preparing the niclosamide microneedle patch according to claim 4, characterized in that: The niclosamide microneedle patch is prepared by the following steps: (1) mixing mesoporous silica nanoparticles and leukocyte / platelet hybrid membranes in a solution, adding near-infrared fluorescent dye and niclosamide, mixing evenly and freeze-drying to obtain composite nanoparticles; (2) mixing the composite nanoparticles obtained in step (1) and the hyaluronic acid solution evenly, placing the mixture in a microneedle mold, and centrifuging the mixture; (3) adding hyaluronic acid solution to the microneedle mold after centrifugation in step (2) for a second time, centrifuging again, and finally vacuum drying to obtain.

6. The method for preparing the niclosamide microneedle patch according to claim 5, characterized in that: In the step (2), the molar volume ratio of the composite nanoparticles to the hyaluronic acid solution is 1-5 μmol: 20-100 μL; and the mass concentration of the hyaluronic acid solution is 13-18%.

7. The method for preparing the niclosamide microneedle patch according to claim 5, characterized in that: The centrifugation conditions in step (2) are: first centrifugation at 3-5°C and 1000-2000 rpm for 5-20 min, and then centrifugation at 3-5°C and 3000-5000 rpm for 5-20 min.

8. The method for preparing the niclosamide microneedle patch according to claim 5, characterized in that: The volume ratio of the hyaluronic acid solution added twice in step (3) to the mixed solution in the microneedle mold in step (2) is 20-40:40-60; the mass concentration of the hyaluronic acid solution is 8-12%.

9. The method for preparing the niclosamide microneedle patch according to claim 5, characterized in that: The conditions for the second centrifugation in step (3) are: centrifugation at 3-5°C and 2000-4000 rpm for 10-30 min.

10. Use of the niclosamide microneedle patch according to claim 4 in the preparation of drugs or medical devices for treating endometriosis.

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