Application of remegapam as medicine for treating adenomyosis

Abstract

The invention relates to a medicine application, in particular to an application of remegapam as a medicine for treating adenomyosis. The invention aims to provide application of remegapam as a medicine for treating adenomyosis, and the medicine can be used for relieving the adenomyosis and improving fertility. According to the technical scheme, the application of the remegapam as the medicine for treating adenomyosis is characterized in that the molecular formula of the remegapam is C28H28F2N6O3, and the molecular weight is 534.557; the CAS (China Area Standard) number is 1289023-67-1; according to the application of the remegapam as the medicine for treating the adenomyosis, the molecular formula of the remegapam is C28H28F2N6O3, and the molecular weight of the remegapam is 534.557; and the CAS number of the strain is 1289023-67-1.

Description

Technical Field

[0001] The present invention relates to a drug application, in particular to the application of Remegepam as a drug for treating adenomyosis. Background Art

[0002] Adenomyosis is a common disease in women of childbearing age in which endometrial glands and stroma invade the myometrium to form diffuse or focal lesions. Its main clinical manifestations are increased menstrual volume and progressive dysmenorrhea, which seriously affect the patient's quality of life and mental health. At the same time, adenomyosis has a negative impact on fertility, including high risks of miscarriage, premature birth, small for gestational age, fetal malformation and postpartum hemorrhage. Even with assisted reproduction, adenomyosis is associated with a lower clinical pregnancy rate and a higher miscarriage rate.

[0003] Existing treatment techniques for adenomyosis include surgery and drugs. Drug treatment mainly includes gonadotropin-releasing hormone analogs (GnRHa) and dienogest. GnRHa can effectively control the progression of lesions, but it cannot be used for a long time. Generally, after 3 injections, patients will have symptoms similar to perimenopausal syndrome and osteoporosis due to severe low estrogen status, and the lesions are prone to recurrence after drug withdrawal; dienogest needs to be taken orally for a long time, which is effective for pain control, but may cause irregular vaginal bleeding and has no significant effect on shrinking the enlarged uterus. For surgical treatment, radical hysterectomy can be selected for adenomyosis, but this is not suitable for women with fertility requirements; adenomyosis lesion resection can also be selected, but because there is no clear boundary between the adenomyosis lesion tissue and the uterine tissue, it cannot be completely removed, and the residual lesions may continue to progress and cause disease recurrence. In addition, women who become pregnant after surgery are at risk of uterine rupture, placenta implantation, and fetal arrest. The above treatment methods have their own side effects, and their treatment effects and application prospects are unsatisfactory. Therefore, there is an urgent need to develop a drug that can reduce patients' pain on the one hand, and alleviate the progression of adenomyosis and improve fertility on the other hand. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide the use of Remigipam as a drug for treating adenomyosis. The drug can alleviate adenomyosis and improve fertility.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Use of remegipam as a drug for treating adenomyosis, wherein the molecular formula of remegipam is C 28 H 28 F 2 N 6 O 3 , molecular weight is 534.557; CAS number is 1289023-67-1.

[0007] The present invention has confirmed in a cell model that remegipam can inhibit the fibroblast-myofibroblast transformation of AMDF; therefore, remegipam can be used as a new drug target for adenomyosis.

[0008] The present invention has confirmed in an animal model that Remigipam can be used to alleviate adenomyosis and improve fertility.

[0009] The beneficial effects of the present invention are:

[0010] Based on the discovery of RAPM1 as a new target for adenomyosis drugs, the present invention confirms that remegipam, which was originally used to treat acute migraine, can be used to treat adenomyosis and improve fertility. Moreover, it does not have the side effects of traditional drugs (GnRHa, dienogest, steroidal anti-inflammatory analgesics) and surgical treatments (radical hysterectomy, lesion resection); therefore, the invention can be used as a new drug for the treatment of adenomyosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a picture of clinical adenomyosis lesions with excessive sensory innervation and fibrosis in the examples.

[0012] Figure 2 Figure 2 is a diagram of sensory hyperinnervation and fibrosis of lesions in the adenomyosis mouse model in the examples.

[0013] Figure 3 This is a graph showing that sensory denervation alleviates adenomyosis fibrosis and lesion infiltration depth in mice in the Examples.

[0014] Figure 4 This is a graph showing the enrichment of RAMP1 gene in the mouse adenomyosis lesion fibroblast population in the example.

[0015] Figure 5 The diagram shows the co-culture of dorsal root ganglion DRG and adenomyosis-derived fibroblasts and the effect of CGRP on promoting myofibroblast transformation of adenomyosis-derived fibroblasts in the examples.

[0016] Figure 6 This is a diagram showing that Remigipam alleviates fibrosis progression and lesion infiltration in mice with adenomyosis in the examples.

[0017] Figure 7 Graph showing the results of the fertility experiment in the examples. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments shown in the accompanying drawings.

[0019] 1. Histological identification of adenomyosis lesions

[0020] After approval by the ethics committee, samples of uterine myometrial lesions from patients with adenomyosis (3 samples) were collected as the experimental group and samples of normal uterine myometrial tissue from patients who underwent hysterectomy for uterine fibroids (3 samples) were collected as the control group. All patients did not receive hormone therapy within 6 months. After fresh tissue collection, it was repeatedly rinsed 3 times with sterile PBS. Protein samples and RNA samples were extracted using RIPA lysis buffer and TRIzol for subsequent testing, and a portion of the tissue was frozen for immunofluorescence and immunohistochemistry detection.

[0021] See also Figure 1 AH diagram:

[0022] Figure 1 Figure A is a picture of the same sample in two groups after being processed by different staining methods. The leftmost column is the pan-neural marker PGP9.5 immunofluorescence staining picture. The strip-like highlights (green) in the picture are PGP9.5 positive areas. Compared with the control group, the PGP9.5 positive density in the adenomyosis group increased significantly, indicating that the nerve density of the adenomyosis lesion increased; the middle column is the HE staining picture. A circle of ectopic lesions can be seen in the adenomyosis group by HE staining, which corresponds to the picture on the left, indicating that the nerves are concentrated around the lesions; the rightmost column is the Masson staining picture. Compared with the control group, the adenomyosis group has a deeper staining, indicating that the ectopic lesions are fibrotic.

[0023] Figure 1 Figure B is the result of quantitative analysis of the PGP9.5 positive staining area in the leftmost column of Figure A; the control group (NC) vs. adenomyosis group (AM) is: 7.79±2.81% vs. 68.77±8.64%. The statistical method used is t test. Among them: * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.001, and the same below.

[0024] Figure 1 Figure C is a quantitative analysis of the collagen fiber staining positive area in the rightmost column of Figure A. The control group vs. adenomyosis is 8.06±1.36% vs. 38.59±5.32%. The statistical method used is t test.

[0025] Figure 1 The highlighted cord-like area in Figure D is the co-stained positive area for the sensory nerve C fiber marker CGRP and the nerve fiber marker TUBB3; compared with the control group, the density of CGRP+ sensory nerves in the adenomyosis group was significantly increased.

[0026] Figure 1Figure E is a bar graph of quantitative analysis of the TUBB3-positive staining area in Figure D; the control group vs. adenomyosis is: 4.38±1.46% vs. 49.40±13.65%. The statistical method used is t-test.

[0027] Figure 1 Figure F is a bar graph of quantitative analysis of the CGRP-positive staining area in Figure D; the control group vs. adenomyosis was 4.13±0.65% vs. 12.52±1.04%. The statistical method used was t-test.

[0028] Figure 1 The G graph shows the protein results; when the internal reference is the same, the adenomyosis group has increased expression of extracellular matrix proteins FN, COL1A1, aSMA, nerve-related indicators NGF, PGP9.5, CGRP and its receptor RAMP1 compared with the control group. In the figure, the grayscale values ​​of the last three samples are much greater than those of the first three samples. This further indicates that there is fibrosis in the adenomyosis lesions and increased expression of nerve-related proteins, suggesting that the progression of adenomyosis is related to nerves.

[0029] Figure 1 Figure H is a quantitative analysis of the expression levels of each gene; the results show that compared with the control group, the gene expression of extracellular matrix proteins FN (1.0±0.09 vs 1.99±0.22), COL1A1 (1.0±0.17 vs 2.09±0.22), ACTA2 (1.0±0.07 vs 1.63±0.11) and nerve-related indicators NGF (1.0±0.13 vs 6.23±0.84), PGP9.5 (1.0±0.09 vs 5.80±0.88), RAMP1 (1.0±0.72 vs 1.28±0.09) in the adenomyosis group increased. The statistical method used was t-test.

[0030] Obviously, Figure 1 Adenomyotic lesions with CGRP+ sensory hyperinnervation and fibrosis are shown.

[0031] 2. Establishment and identification of mouse adenomyosis model and extraction of mouse dorsal root ganglia

[0032] ICR pregnant mice (35-50 g, 17-19 days of gestation) were raised in a specific pathogen-free environment. Tamoxifen citrate tablets were ground into fine powder, and a mixture of peanut oil / lecithin / condensed milk (volume ratio: 2:0.2:3) containing 0.1 mg / ml tamoxifen was prepared. Female newborn mice were drip-fed at a dose of 10 μl / g·bw (body weight) on days 2-5 after birth (the day of birth was the first day); newborn mice in the control group were drip-fed with a solvent without tamoxifen at the same time. Mice were breastfed by mother mice within 3 weeks of birth, separated into cages at 3 weeks, and raised conventionally. Mice were killed on day 42 and bilateral uteri were taken for HE staining and α-SMA&E-CAD immunofluorescence staining for model identification.

[0033] The mice were killed by carbon dioxide and then decapitated. The dorsal skin was cut along the spine to expose the subcutaneous tissue. The scalpel was used to cut open the spinous processes of each vertebra along the spine. The rongeurs were used to remove the spinous processes from top to bottom, and the muscle tissue attached to each spinous process was removed at the same time. The spinal canal was cut from top to bottom with the tip of the rongeurs to expose the spinal cord. Sesame-like white nodules, namely DRG, can be observed at the intervertebral foramen. The finger was used to feel the position at the highest point of the iliac crest, which was used as a bony landmark, corresponding to the L4 dorsal root ganglion. L2-L5 dorsal root ganglia were taken from each mouse, part of which was fixed in 4% paraformaldehyde and then embedded, and part of which was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for WB, co-culture and other experiments.

[0034] See also Figure 2 IP map:

[0035] Figure 2 Figure I is a flowchart of adenomyosis mouse modeling; modeling began on the second day after the mice were born, the mice were separated into cages on the 21st day, the uterus was removed on the 42nd day to observe the modeling situation, and the dorsal root ganglia were taken for characterization.

[0036] Figure 2 Figure J is a picture of the uterus of the control group and the modeling group (adenomyosis group) after different staining treatments. The leftmost column shows the morphology of the mouse uterus under HE staining; compared with the control group, the uterine cavity structure of the mice in the adenomyosis group is irregular, and there is no clear boundary between the endometrium and the myometrium; the middle column is the Masson staining picture, the small picture in the upper right corner is a low-power microscope picture of the uterus stained with Masson, and the main picture is an enlarged picture; compared with the control group, the uterine cavity of the mice in the adenomyosis group has a higher grayscale value, indicating that the degree of fibrosis is more serious; the rightmost column is the co-staining fluorescence image of the epithelial cell indicator E-CAD and the smooth muscle cell indicator aSMA; in the adenomyosis group, the E-CAD-positive area at the triangle mark in the lower left corner can be seen to be wrapped by aSMA, indicating that the gland has invaded the myometrium. In general, Figure 2 The J figure shows that the adenomyosis mouse model was successfully constructed and that the uterus of the model group had severe fibrosis.

[0037] Figure 2Figure K is a bar graph of quantitative analysis of the collagen fiber positive staining area in Masson's staining in Figure J; the control group vs. modeling group was: 13.71±1.84% vs. 48.05±2.98%, and the statistical method used was t-test.

[0038] Figure 2 Figure L is a bar graph of the quantitative analysis of the depth of glandular infiltration in the muscular layer in HE staining in Figure J; the control group vs. modeling group is: Grade 0 vs. Grade 2.92±0.33. The statistical method used is t-test.

[0039] Figure 2 Figure M shows the co-staining of CGRP and TUBB3 in the uterus of 6-week-old mice in the control group and the modeling group; the triangle marks in the left column are the positive staining areas of TUBB3 and CGRP, indicating that sensory nerves are enriched in the uterus of adenomyosis mice. The right column is an enlargement of the left column.

[0040] Figure 2 Figure N shows the staining of CGRP+ neurons in the dorsal root ganglia of mice in the control group and the model group; the white circular highlights in the figure are CGRP-positive areas. Compared with the control group, the expression of CGRP in the dorsal root ganglia of mice in the adenomyosis group was increased.

[0041] Figure 2 Figure O shows the protein results; when the internal reference GAPDH is the same, the adenomyosis model group has increased expression of extracellular matrix proteins FN, aSMA, nerve-related indicators NGF, PGP9.5, and RAMP1 compared with the control group. In the figure, the grayscale values ​​of the last three samples are much greater than the first three samples. This indicates that the adenomyosis lesions of mice also have fibrosis and increased expression of nerve-related proteins.

[0042] Figure 2 The P graph is a quantitative analysis bar graph of the expression level of each gene; the results show that compared with the control group, the gene expression of the extracellular matrix protein FN (0.88±0.03 vs 4.99±0.41), ACTA2 (1.0±0.12 vs 17.05±3.71) and nerve-related indicators NGF (1.11±0.86 vs 2.79±0.27), PGP9.5 (1.0±0.09 vs 1.99±0.22), RAMP1 (1.0±0.13 vs 3.36±1.26) in the adenomyosis group of mice increased. The statistical method used was t-test.

[0043] Obviously, Figure 2 Shows: Mouse adenomyosis lesions present the same characteristics as clinical samples, namely, the presence of CGRP+ sensory nerve enrichment and fibrosis in the lesions.

[0044] 3. Construction of a denervated adenomyosis mouse model

[0045] Resiniferatoxin (RTX) can selectively destroy sensory nerves. It was dissolved in sterile saline with 10% Tween-80 and 10% ethanol. After the adenomyosis mouse model was established, RTX 150 μg / kg was injected intraperitoneally every 5 days starting from the 42nd day to establish a sensory nerve-deprived mouse model. The control group was injected with solvent. The mice were killed on the 63rd day, and the bilateral uterus was removed for tissue morphology, protein level and gene level detection.

[0046] See also Figure 3 AH diagram:

[0047] Figure 3 Figure A is a flow chart of the modeling of adenomyosis mice with sensory nerves removed; after the successful modeling on day 42, intraperitoneal injection of resiniferatoxin was started, and the uterus was removed on day 63 to observe the progression of adenomyosis.

[0048] Figure 3 Figure B is an immunohistochemical staining of the pan-neural marker PGP9.5. The small picture in the upper right corner is a low-power image, and the main picture is an enlarged image. The black strips in the picture are PGP9.5-positive staining areas. Compared with the modeling group, the PGP9.5-positive staining area in the uterus of the denervated group mice was reduced, indicating that the sensory nerves were successfully removed.

[0049] Figure 3 Figure C is a quantitative analysis bar graph of the PGP9.5-positive staining area, which is 3.39±0.22% vs 0.56±0.10% in the modeling group vs the denervation group.

[0050] Figure 3 Figure D is a picture of the two groups of samples after different staining treatments; the leftmost column is the morphology of the uterus of mice in the simple modeling group and the denervation group under HE staining; compared with the denervation group, the uterine cavity structure of the mice in the simple modeling group was irregular, the ectopic lesions protruded into cysts in the upper left corner, and there was no clear boundary between the endometrium and the myometrium. The middle column of pictures is Masson's staining; the small picture in the upper right corner is a low-power microscope picture of the uterus Masson's staining, and the main picture is an enlarged picture; compared with the denervation group, the modeling group has a more serious degree of fibrosis. The rightmost column is a co-stained fluorescence image of the epithelial indicator E-CAD and the muscle fiber indicator aSMA; in the simple modeling group, it can be seen that the E-CAD-positive area is wrapped by aSMA, the myometrium is disordered, and the glands invade the myometrium. In general, Figure 3 Figure D shows that the lesion infiltration depth and lesion fibrosis degree of the denervated group mice were alleviated compared with those of the model group.

[0051] Figure 3 Figure E is a quantitative analysis bar graph of the collagen fiber positive staining area in Masson staining of Figure D; the model group vs. denervation group is: 49.56±4.10% vs. 9.41±1.83%. The statistical method used is t test.

[0052] Figure 3 Figure F is a quantitative analysis bar graph of the depth of glandular infiltration in the muscular layer in HE staining in Figure D; the model group vs. denervation group is: Grade 3.72±0.36 vs. Grade 2.42±0.15. The statistical method used is t test.

[0053] Figure 3 The G graph shows the protein results; when the internal reference is the same, the denervation group has extracellular matrix proteins FN and aSMA, and the nerve-related indicators NGF, PGP9.5, and CGRP receptor RAMP1 expression is reduced compared with the modeling group. The figure shows that the grayscale values ​​of the last three samples are much greater than the first three samples. This indicates that sensory denervation can alleviate the extracellular matrix deposition in the uterus of adenomyosis mice.

[0054] Figure 3 Figure H is a quantitative analysis bar graph of the expression levels of each gene; the results show that compared with the denervation group, the gene expression of mouse uterine extracellular matrix proteins FN (8.30±0.85 vs 1.9±0.29), ACTA2 (17.05±3.7 vs 3.01±0.59), and nerve-related indicators NGF (12.41±2.17 vs 2.64±0.48), PGP9.5 (21.60±5.94 vs 2.2±0.41), RAMP1 (1.98±0.12 vs 0.84±0.12) in the simple modeling group increased. The statistical method used was t-test.

[0055] Obviously, Figure 3 showed that sensory denervation can slow adenomyosis lesion growth and extracellular matrix synthesis.

[0056] 4. Single-cell sequencing analysis and histological identification of adenomyosis lesions

[0057] Uterine myometrial lesion samples were collected from 3 patients with adenomyosis as the experimental group, and uterine normal myometrial tissue samples were collected from 3 patients who underwent hysterectomy for uterine fibroids as the control group.

[0058] Six muscle tissue samples for single-cell sequencing were digested with digestion solution at 37°C in a shaker to make single-cell suspensions. After sieving and lysing, the single-cell suspensions were quality-checked and counted. The cell survival rate was above 80%. The qualified cells were washed and resuspended to a suitable cell concentration of 700-1200 cells / ul for 10xGenomics Chromium TMThe system was operated on the machine. The cell viability of the NC group samples used for scRNA sequencing was 97%, 87%, and 90%, respectively, and the cell viability of the AMWD group samples was 83%, 88%, and 89%, respectively, which met the quality inspection requirements.

[0059] Seurat software was used for further quality control filtering to remove unqualified data such as double cells, dead cells, and cell fragments, and to enrich the first-strand cDNA, followed by cDNA amplification and quality inspection. The cDNA that passed the quality inspection was used to construct the second-generation sequencing library, and after fragmentation, connection of sequencing adapters, sample IndexPCR and other experimental processes, the library was finally quantitatively inspected. The constructed library was sequenced using the Illumina NovaSeq6000 platform and the PE150 sequencing mode, with a sequencing volume of 20k reads / cell and above. After obtaining the sequencing data, we used the 10X Genomics official software Cell Ranger v7.0.0 to quality control the raw data. Seurat software was used for further quality control filtering to remove unqualified data such as double cells, dead cells, and cell fragments.

[0060] In order to identify the main cell clusters, UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction cluster analysis was performed using the R software package Seurat, and the software SingleR was used for cell type annotation.

[0061] Figure 4 Figure A shows the distribution of various cell populations in single-cell sequencing;

[0062] Figure 4 Figure B shows the result of RAMP1 gene staining in single-cell sequencing. The brighter areas in the figure are the cells enriched with RAMP1. Combining the two figures, we can see that the CGRP receptor RAMP1 is mainly distributed in the fibroblast population, so we extracted fibroblasts from adenomyosis lesions for subsequent cell experiments.

[0063] 4.1 Isolation and culture of primary adenomyosis-derived fibroblasts (AMDFs)

[0064] After approval by the Ethics Committee, adenomyosis lesion tissue was obtained under sterile conditions, placed in a 6cm cell culture dish, and rinsed with PBS three times. The tissue was cut into 0.5mm3 tissue blocks with sterile ophthalmic scissors, transferred to a 15ml centrifuge tube, 3 volumes of DMEM / F12 medium were added, and type IV collagenase (4mg / ml) was added according to the final volume, and placed in a 37°C shaker for digestion for 1 hour. The digested tissue fluid was filtered using 100μm and 40μm cell strainers, the filtrate was collected, centrifuged at 1200rpm for 10 minutes, the supernatant was discarded, resuspended with DMEM / F12 complete medium, counted, plated, and cultured in a 37°C, 5% CO2 cell culture incubator. After that, the medium was changed every 2 days, and the cell density under the microscope reached about 80% when the passage was subcultured. At the same time, the cultured AMDF was identified by immunofluorescence double staining with rabbit anti-human vimentin antibody VIM and mouse anti-human α-SMA.

[0065] 4.2 Detection of the effect of CGRP on fibroblasts derived from human adenomyosis lesions (see Figure 5 AO Figure) AMDF suspension was inoculated in a 12-well plate, with a cell volume of 5×10 5 The cells were divided into three groups: control group, CGRP group, and remegipam group. The 12-well plate was placed in an incubator for pre-culture for 3 hours. After the cells adhered to the wall, the medium was changed to ensure that the volume of the culture medium in each well was consistent. CGRP and remegipam were added to the experimental groups respectively. After 6 hours, CGRP was added to the remegipam group. After 48 hours of culture, the cells were collected, and protein and RNA were extracted for biological detection.

[0066] Figure 5 Figure A is a schematic diagram of the in vitro co-culture of rat dorsal root ganglion cells and adenomyosis lesion-derived fibroblasts.

[0067] Figure 5 Figure B is a co-staining of aSMA and VIM immunofluorescence in fibroblasts; the left side is the control group, the middle is the cells co-cultured with dorsal root ganglia, and the right side is the cells co-cultured with dorsal root ganglia + treated with CGRP receptor blocker Remigipam; the figure shows that the expression of extracellular matrix protein aSMA increased after co-culture with dorsal root ganglia, and decreased after adding Remigipam. This suggests that dorsal root ganglia can promote the transformation of fibroblasts derived from lesions into myofibroblasts, and this phenomenon can be inhibited after adding CGRP receptor blocker Remigipam.

[0068] Figure 5Figure C is a quantitative analysis bar graph of aSMA-positive staining areas in Figure B; control vs DRG vs DRG+Rime: 17.94±1.70% vs 44.41±3.88% vs 19.24±3.95%, and the statistical methods used were ANOVA and Tukey multiple comparisons. In the figure: control is the AMDF control group, DRG is the AMDF group under DRG co-culture, and DRG+Rime is the AMDF group with DRG co-culture and the addition of Remigipam, and the same below.

[0069] Figure 5 Figure D shows the protein results; when the internal reference is the same, the DRG co-culture group has increased expression of extracellular matrix proteins FN and aSMA compared with the control group, while the expression decreases after the addition of remegipam, which is shown in the figure as the gray value of the middle sample is much greater than the left and right samples. This indicates that the dorsal root ganglion can promote the transformation of lesion-derived fibroblasts into myofibroblasts, and this phenomenon can be inhibited after the addition of the CGRP receptor blocker remegipam.

[0070] Figure 5 Figure E is a quantitative analysis bar graph of FN and aSMA gene expression levels; the results show that after co-culture, the gene expression levels of extracellular matrix proteins FN (control vs DRG vs DRG+Rime: 1.00±0.03 vs 1.72±0.04 vs 1.31±0.06) and aSMA (control vs DRG vs DRG+Rime: 1.00±0.11 vs 1.13±0.07 vs 0.89±0.02) increased. The statistical methods used were ANOVA and Tukey multiple comparisons.

[0071] Figure 5 Figure F shows the result of Tunel apoptosis staining. The highlighted area in the figure is the positive area of ​​apoptosis staining. Compared with the control group, co-culture of dorsal root ganglia reduced fibroblast apoptosis, which can be inhibited by remegipam.

[0072] Figure 5 Figure G is a quantitative analysis bar graph of Tunel apoptosis staining results, control vs DRG vs DRG+Rime: 50.39±8.09% vs 2.48±1.75% vs 20.84±3.94%. The statistical methods used were ANOVA and Tukey multiple comparisons.

[0073] Figure 5 Figure H is a schematic diagram of CGRP directly acting on fibroblasts derived from adenomyosis lesions.

[0074] Figure 5Figure J is a co-staining of aSMA and VIM immunofluorescence in fibroblasts; the left side is the control group, the middle is the CGRP-treated picture, and the right side is the CGRP+Remedipam-treated picture. The figure shows that the expression of the extracellular matrix protein aSMA increased after CGRP treatment, and decreased after the addition of Remedipam. This suggests that CGRP can promote the transformation of lesion-derived fibroblasts into myofibroblasts, and this phenomenon can be inhibited after the addition of the CGRP receptor blocker Remedipam.

[0075] Figure 5 Figure K is a quantitative analysis bar graph of the aSMA-positive staining area in Figure J; NC vs CGRP vs C+Rime is: 9.40±2.34% vs 23.20±1.77% vs 6.40±2.54%; the statistical methods used are ANOVA and Tukey multiple comparisons.

[0076] Figure 5 Figure L shows the protein results; when the internal reference GAPDH is equal, the CGRP group has increased expression of extracellular matrix proteins FN and aSMA compared with the control group; and the expression decreases after the addition of remegipam, which is shown in the figure as the gray value of the middle sample is much greater than the left and right samples. This indicates that CGRP can promote the transformation of fibroblasts from lesions into myofibroblasts, and this phenomenon can be inhibited after the addition of remegipam.

[0077] Figure 5 Figure M is a quantitative analysis bar graph of FN and aSMA gene expression levels; the results show that after CGRP treatment, the gene expression levels of extracellular matrix proteins FN (control vs CGRP vs C+Rime: 1.00±0.04 vs 1.52±0.07 vs 1.33±0.09) and aSMA (control vs CGRP vs C+Rime: 1.00±0.02 vs 1.28±0.07 vs 1.05±0.06) increased. The statistical methods used were ANOVA and Tukey multiple comparisons.

[0078] Figure 5 Figure N shows the result of Tunel apoptosis staining, and the highlighted area in the figure is the positive area of ​​apoptosis staining; compared with the control group, CGRP treatment reduced fibroblast apoptosis, and this phenomenon can be inhibited by Remigipam.

[0079] Figure 5Figure O is a quantitative analysis bar graph of Tunel apoptosis staining; control vs CGRP vs C+Rime is 46.80±9.94% vs 1.37±0.53% vs 29.75±5.26%. The statistical methods used are ANOVA and Tukey multiple comparisons.

[0080] Obviously, Figure 5 It was shown that CGRP promoted the differentiation of adenomyosis lesion-derived fibroblasts into myofibroblasts, and the CGRP receptor antagonist remegipam could inhibit this phenomenon.

[0081] 5. Verification of the therapeutic effect of Remigipam on mouse adenomyosis model (see Figure 6 AG diagram)

[0082] Figure 6 Figure A is a flow chart of the intervention of adenomyosis mice with Remigipam; referring to the dosage of Remigipam in animal experiments and the conversion of human-mouse equivalent doses in the literature, the oral dose of the blocker for mice in this experiment was 10 mg / kg·bw. When the modeling mice were conventionally raised to 42 days of age, the mice were randomly divided into two groups, namely the modeling group and the Remigipam group, with 10 mice in each group. Remigipam was prepared using sodium carboxymethylcellulose (CMC-Na) to make a drug suspension, which was administered by gavage. The model group was gavaged with CMC-Na under the same weight standard. Three weeks after administration, a mouse hot plate experiment was performed to observe changes in the thermal pain sensitivity of mice. The specific operation of the hot plate experiment is: after taking out the mice, let the mice adapt to the test room for 15-30 minutes. Set the temperature of the instrument hot plate to 55 degrees, put the animals on the hot plate and start timing immediately, observe the time when the animals begin to retract or lick their hind feet, set the end time to 60s to avoid burns to the mice, and repeat the experiment at an interval of 1h. After the experiment, the mice were killed and the gross morphology of the uterus was observed. The specimen was divided into two parts. One part was fixed with 4% paraformaldehyde and then embedded in paraffin for sectioning. HE staining, Masson staining and other tissue morphology observations were performed. The other part was quickly frozen in liquid nitrogen and stored at -80°C. Tissue RNA and protein were extracted for molecular biology experiments.

[0083] Figure 6 Figure B is a picture of the three groups of samples after different staining treatments. The top row is HE staining; the bottom row is Masson staining, the small picture in the upper right corner is a low-power microscope picture of the uterus Masson staining, and the main picture is an enlarged picture. HE showed that compared with the control group, the uterine cavity structure of the modeling group mice was irregular, large ectopic lesions were visible above, and there was no clear boundary between the endometrium and the myometrium; the depth of lesion infiltration in the mice in the Remigipam group was alleviated compared with the modeling group; Masson staining showed that the gray value of the uterus of the modeling group mice was higher than that of the control group and the intervention group, which indicated that the degree of fibrosis of the lesions in the intervention group was alleviated compared with the modeling group.

[0084] Figure 6 Figure C is a quantitative analysis bar graph of the collagen fiber positive staining area in Masson staining in Figure B; the control group vs the model group vs the remegipam group are: 13.32±1.94% vs 48.91±3.08% vs 20.67±2.58%. The statistical methods used were ANOVA and Tukey multiple comparisons.

[0085] Figure 6 Figure D is a quantitative analysis bar graph of the depth of glandular invasion in the muscular layer in HE staining in Figure B; the control group vs the model group vs the remegipam group is: 0 vs 3.92±0.31% vs 2.79±0.37. The statistical methods used were ANOVA and Tukey multiple comparisons.

[0086] Figure 6 Figure E is a quantitative analysis bar graph of the mouse hot plate test; the mice in the Remgipam group had lower thermal sensitivity than the mice in the modeling group (7.23±0.71s vs 4.74±0.37s), which means that adenomyosis can reduce the thermal pain threshold of mice, and Remgipam has an analgesic effect (7.07±0.66s), which can relieve the discomfort caused by the disease. The statistical methods used were ANOVA and Tukey's multiple comparisons.

[0087] Figure 6 Figure F shows the protein results; although the gray value of the internal reference GAPDH in the modeling group is lower than that in the control group, the expression of extracellular matrix proteins FN and COL1A1, nerve-related indicators NGF, PGP9.5, and CGRP receptor RAMP1 in the Remgipam group is lower than that in the modeling group, as shown in the figure, the gray values ​​of the last three samples are much lower than the three samples in the middle. This indicates that Remgipam can alleviate the extracellular matrix deposition in the uterus of adenomyosis mice.

[0088] Figure 6 Figure G is a quantitative analysis bar graph of the expression levels of each gene; the results showed that compared with the other two groups, the gene expression of uterine extracellular matrix proteins FN (1.00±0.13 vs 3.62±0.62 vs 0.48±0.09), ACTA2 (1.00±0.14 vs 2.47±0.23 vs 0.39±0.06) and nerve-related indicators NGF (1.00±0.13 vs 2.47±0.47 vs 1.10±0.27), PGP9.5 (1.00±0.21 vs 3.56±0.94 vs 1.32±0.46), and RAMP1 (1.00±0.20 vs 1.32±0.42 vs 0.83±0.18) in the mice in the remgipam intervention group was decreased.

[0089] Obviously, Figure 6Results showed that Remigipam could alleviate the progression of fibrosis and lesion infiltration and relieve pain in mice with adenomyosis.

[0090] 6. Fertility assessment (see Figure 7 AB diagram)

[0091] Female mice were caged with male mice at a ratio of 2:1. After caged, the female mice were checked for vaginal plugs at 6-8 o'clock every day. The plug was counted as 0.5 days. The mice were killed on the 4.5th day to remove the uterus. The Bruce blue test was used to observe the number of pregnant uteri and the number of embryos implanted in each uterus.

[0092] Figure 7 Figure A is a mouse fertility experiment. The "V" shape in the figure is the mouse uterus, and the protruding parts of the uterus in the left and right figures are embryos. Compared with the modeling group, the pregnancy rate of adenomyosis mice decreased, and the pregnancy rate increased after intervention with Remigipam.

[0093] Figure 7 Figure B shows the embryonic status of mice in the modeling group and the intervention group on the 16th day of pregnancy. The outlines of the embryos and placentas of mice in the modeling group are not clear, and some embryos have been aborted and absorbed, while the outlines of the embryos and placentas of mice in the intervention group are clear. The above results show that Remgipam can effectively improve the fertility level of mice with adenomyosis.

[0094] The quantitative analysis results of the pregnancy rate and abortion rate per uterus of each group are shown in Table 1. The table shows that compared with the modeling group, the uterine pregnancy rate of mice in the intervention group increased (37.9% vs 64.7%), and the embryo abortion rate decreased (21.8% vs 14.5%).

[0095] Table 1. Fertility results of adenomyosis mouse model after different treatments

[0096]

[0097] a) p<0.01 model vs control

[0098] b) p<0.01 model vs control

[0099] c) p<0.01Remigipam vs modeling

[0100] Apparently, Remigipam can improve the fertility rate and reduce the abortion rate in adenomyosis mice.

[0101] In summary: Rimegepant is a RAMP1 antagonist with a molecular formula of C 28 H 28 F 2 N 6 O 3; is a drug for the treatment of migraine approved by the State Administration for Market Regulation. The present invention confirms that oral administration of remegipam can reverse fibrosis, reduce thermal pain sensitivity, and improve fertility in a mouse adenomyosis model; in short, the applicant has found that remegipam can be used as a new drug target for adenomyosis and can be used as a drug for the treatment of adenomyosis.

Claims

1. Use of Remigipam as a drug for treating adenomyosis, wherein the molecular formula of Remigipam is C 28 H 28 F2N6O3, molecular weight is 534.557; CAS number is 1289023-67-1.

2. The use of Remegepam as a drug for treating adenomyosis according to claim 1, characterized in that: The remegipam can inhibit the fibroblast-myofibroblast transformation of AMDF; therefore, remegipam can be used as a new drug target for adenomyosis.

3. The use of Remegepam as a drug for treating adenomyosis according to claim 2, characterized in that: Remeditate can be used to relieve adenomyosis and improve fertility.

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