Use of dasatinib for treating endometriosis

Abstract

The invention discloses an application of dasatinib in preparation of a medicine for treating endometriosis. The dasatinib can be used for treating endometriosis. The new application of the dasatinib in treating the endometriosis has important clinical significance on accurate treatment of the endometriosis.

Description

Technical Field

[0001] The present invention relates to a new use of dasatinib, and in particular to the use of dasatinib for treating endometriosis. Background Art

[0002] Endometriosis is a gynecological disease characterized by the colonization, growth, and infiltration of endometrial-like tissue outside the uterine cavity. At present, the relevant theories of the pathogenesis of endometriosis mainly include the retrograde menstrual theory, the implantation of endometrial stem cells, and the metaplasia of the coelomic epithelium. In addition, there are also inflammatory factors, immune regulation and hormonal abnormalities, environmental factors, genetic and epigenetic factors involved. In terms of the pathogenesis, the current drugs for the treatment of endometriosis include traditional drugs and emerging drugs. Traditional drugs are based on inhibiting ovulation, blocking menstruation, and maintaining a stable endocrine hormone environment, including: ① gonadotropin-releasing hormone agonists that create a low estrogen environment; ② danazol and gestrinone that create a high androgen environment; ③ oral contraceptives and progesterone that create a high progesterone environment. However, these drugs only temporarily relieve symptoms, and the lesions resume metabolic activity after drug treatment is stopped, and these drugs are not suitable for women who need natural conception. Emerging drugs mainly include: ① anti-angiogenic drugs: such as TNP-470; ② drugs that inhibit cell proliferation: such as statins; ③ demethylation drugs and histone deacetylase inhibitors; ④ anti-inflammatory drugs and immunomodulators. Most of the emerging drugs are still in the exploratory stage, and these drugs will damage the normal endometrium while inhibiting the ectopic endometrium, which limits their application in the treatment of endometriosis. Therefore, there is an urgent need to develop drugs that target the inhibition of ectopic endometrium in clinical practice, which has important clinical significance for the precise treatment of endometriosis. Summary of the invention

[0003] The object of the present invention is to provide a new use of dasatinib for treating endometriosis.

[0004] In one embodiment, the dasatinib selectively inhibits the growth of human endometriosis cells.

[0005] In one embodiment, the dasatinib selectively inhibits the growth of stromal cells and epithelial organoids from patients with endometriosis.

[0006] In one embodiment, the dasatinib selectively induces apoptosis of ectopic endometrial cells.

[0007] In one embodiment, the dasatinib partially inhibits the growth of ectopic endometrium.

[0008] The new use of dasatinib for treating endometriosis provided by the present invention has important clinical significance for the precise treatment of endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1A and Figure 1B The results show that dasatinib of Example 1 selectively inhibits the growth of immortalized human endometriosis cells 12Z;

[0010] FIG. 2A to FIG. 2D The results show that dasatinib of Example 2 can selectively inhibit the growth of primary stromal cells and epithelial organoids from patients with endometriosis;

[0011] FIG. 3A to FIG. 3D The results show that dasatinib in Example 3 can selectively induce apoptosis of ectopic endometrial cells;

[0012] FIG. 4A to FIG. 4D The results show that dasatinib of Example 4 can partially inhibit the growth of ectopic endometrium;

[0013] Figure 5A and Figure 5B The results show that dasatinib of Example 5 can partially inhibit the growth of ectopic endometrium. DETAILED DESCRIPTION

[0014] Endometriosis and malignant tumors have some similarities in some aspects, including that both cells are highly invasive and metastatic, and ectopic endometrial cells are prone to mutations in tumor-related genes (Artemova D, Vishnyakova P, Khashchenko E, et al. Endometriosis and Cancer: Exploring the Role of Macrophages [J]. International Journal of Molecular Sciences, 2021), but they are two completely different diseases. According to statistics, the probability of endometriosis becoming cancerous is generally a few thousandths, so the probability of cancer is very small. Endometriosis is a benign disease, while malignant tumors are invasive tumors formed by abnormally proliferating cells. The treatment methods and prognosis of the two are also completely different. Therefore, even if there are similarities in some aspects, their essences are still different.

[0015] The applicant applied drug screening technology for the first time, selected a targeted therapeutic drug library (MCE, HY-L080) for flux screening in normal endometrial cell lines (hESC) and immortalized human endometriosis cells (12Z), and obtained dasatinib. This application breaks through the limitations of previous research in terms of ideas and methods, keeps up with the current concept of precise disease treatment, and provides a new theoretical basis for the development and research of drugs for the precise treatment of endometriosis.

[0016] Dasatinib

[0017] Dasatinib is an oral tyrosine kinase inhibitor that inhibits the activity of the BCR-ABL fusion protein. Its main indications include chronic myeloid leukemia (CML), especially patients who are resistant or intolerant to other treatments (such as imatinib); acute lymphoblastic leukemia (ALL), for patients with BCR-ABL-positive acute lymphoblastic leukemia. The main target of dasatinib is the BCR-ABL tyrosine kinase, a fusion protein formed by a translocation between chromosomes 9 and 22. This fusion protein plays a key role in CML and certain types of ALL, promoting the proliferation and survival of cancer cells. By inhibiting the activity of BCR-ABL, dasatinib can effectively reduce the proliferation of cancer cells and induce their apoptosis. In addition, dasatinib also has an inhibitory effect on some other tyrosine kinases (such as SRC family kinases), which may also contribute to its anti-tumor effect. The theoretical range of dasatinib's active concentration is from tens of nM to hundreds of nM, and the specific concentration range may vary depending on the cell line and experimental conditions; in clinical use, the recommended dose of dasatinib is 100 mg once daily (for CML patients), which usually corresponds to a plasma concentration of 50-300 nM. This range is considered to be the preferred range that can effectively inhibit BCR-ABL activity and control the disease; based on clinical studies and pharmacokinetic data, the optimal plasma concentration of dasatinib is generally considered to be 100 to 200 nM. This concentration range can achieve good therapeutic effects in most patients while reducing the risk of side effects.

[0018] The present invention provides a new use of dasatinib for treating endometriosis.

[0019] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Example 1

[0021] 2000 normal human endometrial cell line hESC and 2000 immortalized human endometriosis cell line 12Z were inoculated in 96-well plates. After the cells adhered to the plate the next day, they were treated with 9 concentration gradients of dasatinib (two-fold dilutions from 20 μM to 0.078 μM). After 96 hours, the cell proliferation activity was analyzed by CCK8 assay and the IC50 values ​​of the cells were compared.

[0022] Experimental results: The IC50 value of 12Z cells was 0.7594 μM, while that of hESC cells was 6.777 μM. The IC50 of hESC cells was about 8.9 times higher than that of 12Z cells (e.g. Figure 1A as shown).

[0023] Inoculate 1×10 5 hESC cells, 1×10 5 12Z cells were plated in 12-well plates. After the cells adhered to the plate the next day, they were treated with 0.5 μM dasatinib. After 72 hours, the cells were photographed under white light.

[0024] Experimental results: The number of 12Z cells treated with 0.5 μM dasatinib was significantly reduced compared with the number of hESC cells treated with 0.5 μM dasatinib (e.g. Figure 1B as shown).

[0025] The results suggest that dasatinib can selectively inhibit the growth of ectopic endometrial cells.

[0026] Example 2

[0027] Stromal cells were extracted from normal endometrium (ESC_Eutopic) and ectopic endometrial tissue (ESC_Ectopic) of patients with endometriosis (Stem Cell Res Ther. 2017 Nov 7;8(1):251).

[0028] 1×10 interstitial cells were inoculated 5 ESC_Eutopic and 1×10 5 ESC_Ectopic were grown in a 12-well plate and treated with 0.5 μM and 1 μM dasatinib, respectively. After 72 h, the cells were photographed under white light.

[0029] Experimental results: The number of interstitial cells ESC_Ectopic treated with dasatinib was significantly reduced compared with the number of interstitial cells ESC_Eutopic treated with dasatinib ( Figure 2A ).

[0030] 1×10 interstitial cells were inoculated 5 ESC_Eutopic and 1×10 5ESC_Ectopic cells were cultured in a 12-well plate and treated with 9 concentration gradients of dasatinib (from 20 μM to 0.078 μM, diluted two-fold in sequence). After 72 h, the cell viability was detected by CCK8 method, and the IC50 values ​​of interstitial cells ESC_Eutopic and ESC_Ectopic were compared.

[0031] Experimental results: The proliferation level of interstitial cells ESC_Ectopic treated with 0.5 μM and 1 μM dasatinib was significantly reduced compared with that of interstitial cells ESC_Eutopic treated with 0.5 μM and 1 μM dasatinib ( Figure 2B ).

[0032] Organoids of normal endometrium (OVE_EUO) and ectopic endometrium (OVE_ECO) were extracted from patients with endometriosis (Nat Cell Biol. 2019 Aug;21(8):1041-1051).

[0033] The organoids OVE_EUO and OVE_ECO were inoculated in 24-well plates and treated with 0.1 μM and 0.5 μM dasatinib for 5 days, respectively. The images were taken with white light using an EVOS microscope, and CellTiter-Glo was used to detect cell viability.

[0034] Experimental results: The organoids OVE_ECO treated with 0.1 μM and 0.5 μM dasatinib were shrunken or disintegrated compared with the organoids OVE_EUO treated with 0.1 μM and 0.5 μM dasatinib ( Figure 2C ), decreased cell viability ( Figure 2D ).

[0035] The results suggest that dasatinib can selectively inhibit the growth of stromal cells and epithelial organoids from patients with endometriosis.

[0036] Example 3

[0037] Inoculate 1×10 5 hESC cells and 1×10 5 12Z cells were plated in 6-well plates and treated with 0.5 μM dasatinib for 72 h after 24 h. The cells were collected and Annexin V-FITC / PI was detected using flow cytometry. The percentages of various types of cells were calculated to analyze the effect of dasatinib treatment on cell apoptosis.

[0038] Experimental results: After dasatinib treatment, the percentage of apoptotic cells in 12Z cells was 25.3%, which was significantly higher than that of hESC cells (5.9%). Figure 3A and Figure 3B ).

[0039] 1×10 interstitial cells were inoculated 5 ESC_Eutopic and 1×10 5 ESC_Ectopic were plated in 6-well plates and treated with 1 μM dasatinib for 72 h after 24 h. The cells were collected and Annexin V-FITC / PI was detected using flow cytometry. The percentages of various types of cells were calculated to analyze the effect of dasatinib treatment on cell apoptosis.

[0040] Experimental results: After dasatinib treatment, the percentage of apoptotic cells in the interstitial cells ESC_Ectopic was 13.3%, which was significantly increased compared with the percentage of apoptotic cells in the interstitial cells ESC_Eutopic (2.62%). Figure 3C and Figure 3D ).

[0041] These results suggest that dasatinib can selectively induce apoptosis of ectopic endometrial cells.

[0042] Example 4

[0043] Eighteen 8-week-old female NSG immunodeficient mice were prepared. Ectopic endometrial tissue was obtained from patients with endometriosis, cut into 1-2 mm³ fragments with culture medium, and 300 μL was injected subcutaneously for transplantation. Estradiol benzoate (100 μL, 1 mg / mL) was given twice a week for 2 weeks. The mice were divided into three groups. Dasatinib 10 mg / kg group: 10 mg / kg dasatinib was injected intraperitoneally for 15 days starting 5 days after transplantation; Dasatinib 20 mg / kg group: 20 mg / kg dasatinib was injected intraperitoneally for 15 days starting 5 days after transplantation; Vehicle group: 5% DMSO, 40% PEG300, 5% Tween 80 and 50% saline. The weight of mice and the volume of subcutaneous tissue were checked regularly. Samples were taken after the end of administration, and the tissue weight was weighed. Figure 4A A flow chart of animal experiments is shown.

[0044] Experimental results: After 10 mg / kg and 20 mg / kg dasatinib treatment, the volume of ectopic endometrial tissue was significantly reduced compared with vehicle treatment ( Figure 4B After 10 mg / kg and 20 mg / kg dasatinib treatment, the weight of ectopic endometrial tissue was significantly reduced compared with vehicle treatment ( Figure 4C After 10 mg / kg and 20 mg / kg dasatinib treatment, the weight of mice did not change significantly compared with vehicle treatment ( Figure 4D ).

[0045] The results showed that dasatinib could partially inhibit the growth of ectopic endometrium in the human ectopic endometrium mouse transplantation model.

[0046] Example 5

[0047] Twelve 8-week-old BALB / c female mice weighing 15-17 g were prepared and randomly divided into two groups: donor mice group and recipient mice group, 6 mice in each group. Donor mice were used to provide uterine horns to produce endometriosis-like lesions. Donor mice were gavaged with 56 µg / kg / d 17β-estradiol daily for 3 consecutive days. The donor mice were cervically dislocated, and the uterine horns were removed and transferred to a culture dish containing 37°C warm sterile PBS. The uterine horns were opened longitudinally with microscissors and cut into two halves, and each uterus provided 4 samples ready for transplantation into the abdominal cavity of recipient mice. After anesthesia, the recipient mice were incised at the midline of the abdomen, and the samples were sutured to the peritoneal parietal bone with two 7 / 0 polypropylene needles, one sample on the right side and the other sample on the left side. In all mice, the samples were sutured at the same position of the abdominal wall to ensure that the host tissue site showed similar vascularization. The wound was sutured with 6 / 0 nylon suture. All recipient mice were gavaged with 56 µg / kg / day of 17β-estradiol for 3 consecutive days after surgery, and then divided into two groups. One group was intraperitoneally injected with 20 mg / kg dasatinib once a day for 28 days, and the other group was not given any medication. After the end of the experiment, the ectopic endometrial tissue of the mice was collected and weighed (e.g. Figure 5A as shown).

[0048] Experimental results: The average weight of ectopic endometrium in mice treated with 20 mg / kg dasatinib was 55.8 mg, which was significantly lower than the average weight of ectopic endometrium in mice not treated with 20 mg / kg dasatinib (35.8 mg). Figure 5B ).

[0049] The results showed that dasatinib could partially inhibit the growth of ectopic endometrium in the mouse autologous endometrial transplantation model.

[0050] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of dasatinib in the preparation of a medicament for treating endometriosis.

2. The use according to claim 1, characterized in that The dasatinib selectively inhibits the growth of human endometriosis cells.

3. The use according to claim 1, characterized in that The dasatinib selectively inhibits the growth of stromal cells and epithelial organoids from patients with endometriosis.

4. The use according to claim 1, characterized in that The dasatinib selectively induces apoptosis of ectopic endometrial cells.

5. The use according to claim 1, characterized in that: The dasatinib partially inhibits the growth of ectopic endometrium.

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