New drug uses of rotigotine

Abstract

The present invention relates to the field of pharmaceutical research and development technology, and specifically to a new drug use of rotigotine. The present invention constructs a Crohn's disease model rat, the model rat ileum mucosal damage, designs a control experiment, and intraperitoneally injects rotigotine (RTG) into the treatment group, and evaluates the disease activity index (DAI), ileum gross injury index (MDI), and ileum histological injury index (TDI) of each group of rats. The results show that chronic treatment with RTG can significantly improve the disease activity score, alleviate ileum histological damage, and inhibit intestinal mucosal inflammation, indicating that RTG can effectively inhibit ileum mucosal damage, promote ileum ulcer repair, inhibit the increase of proinflammatory factors, and significantly improve the symptoms of Crohn's disease. It shows that it is feasible to expand the drug use of RTG to the treatment of Crohn's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical research and development, and in particular to a new pharmaceutical use of rotigotine. Background Art

[0002] The ileum refers to a section of the small intestine that connects the jejunum and the cecum. It is curved and located behind the duodenum and jejunum in the small intestine, followed by the large intestine. The jejunum and ileum are attached to the posterior abdominal wall by the mesentery. The edge with mesentery is called the mesenteric edge, and the opposite edge is called the free edge or anti-mesenteric edge. Crohn's disease (CD) is a chronic nonspecific gastrointestinal inflammatory disease that often occurs in the terminal ileum. Inflammation and ulceration of the intestinal mucosa in this area lead to a series of clinical symptoms, such as abdominal pain and diarrhea. Intestinal mucosal inflammation induced by immune, environmental and genetic factors is considered to be a key link in the onset of CD, but the specific mechanism is still unclear. In recent years, the incidence of CD in my country has increased year by year and is showing a trend of younger age. Clinical manifestations include abdominal pain, diarrhea and weight loss. The disease is prolonged and unresolved, which seriously affects the quality of life of patients, but there is currently no effective cure. Therefore, it is of great significance to explore intervention and treatment strategies for CD-induced inflammation of the terminal ileum.

[0003] Dopamine (DA) is a catecholamine neurotransmitter, mainly produced by the nigrostriatal system, and plays an important role in regulating motor behavior, reward, cognition, sleep, growth and development. DA receptors (DR) can be divided into five subtypes (D1R-D5R). Dysfunction of the DA system is closely related to depression, addiction, schizophrenia and Parkinson's disease. Outside the central nervous system, non-neuronal cells such as chromaffin cells, immune cells and gastric parietal cells in the adrenal medulla can also synthesize, store and release DA. Recent studies have found that DA can promote the ubiquitination of inflammasomes through D1R on macrophages, thereby inhibiting inflammatory responses; it can also inhibit inflammatory responses in the brain through the D2R on astrocytes in the brain and the αB-crystallin pathway. The above studies suggest that DA has systemic and local anti-inflammatory effects.

[0004] DA synthesized in the intestine accounts for more than 50% of the total body synthesis, and the content of DA in the intestinal mucosal layer is twice that of the muscular layer. Recent studies have found that DA in the intestinal lumen can inhibit the secretion of IL-4 and IFN-γ through the D1R of natural killer T cells in the liver, successfully alleviating autoimmune hepatitis in mice. DA in the intestinal mucosal layer can also act on the D5R on macrophages in the colonic lamina propria, promoting their polarization to the anti-inflammatory M2 type and inhibiting their polarization to the pro-inflammatory M1 type, thereby inhibiting colon mucosal inflammation in ulcerative colitis model mice and promoting the remission of the disease. These studies suggest that DA in the intestinal mucosal layer is involved in maintaining local intestinal immune homeostasis. However, ulcerative colitis mainly affects the rectum and part of the colon, and CD mainly affects the mucosal layer of the terminal ileum and ascending colon. Whether DA has a therapeutic effect on CD-induced mucosal damage in the terminal ileum is still lacking.

[0005] Rotigotine (RTG) is a non-specific dopamine agonist that mainly acts on D1R, D2R and D3R in the body. Current studies have shown that RTG can not only be used to treat Parkinson's disease, but also to improve the symptoms of restless legs syndrome. However, the repair effect of RTG on terminal ileal mucosal damage, especially whether it has a therapeutic effect on Crohn's disease, has not been reported yet. Summary of the invention

[0006] In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a new pharmaceutical use of rotigotine, which is applied to the treatment of ileal mucosal damage, especially damage caused by inflammation of the terminal ileum caused by Crohn's disease, and has obvious therapeutic effects on symptom relief and ulcer repair.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] Use of rotigotine (RTG) in preparing a medicine for treating ileal mucosal damage.

[0009] Specifically, the ileal mucosal injury is injury caused by inflammation of the terminal ileum caused by Crohn's disease.

[0010] The embodiment of the present invention verifies that by constructing a Crohn's disease model rat, the rat disease activity index and the gross ileum damage index are used as evaluation indicators, the histological damage index of the terminal ileum is evaluated by immunohistochemical staining, and the levels of proinflammatory factors TNF-α, IL-1β and IL-6 are detected. It is proved that for Crohn's disease model rats with inflammatory damage in the terminal ileum, after the administration of RTG (at a dose of 10 mg / kg), ulcer repair can be promoted, the increase of proinflammatory factors can be inhibited, and the symptoms of Crohn's disease model rats can be significantly improved.

[0011] It should be understood that RTG is used to prepare drugs for treating ileal mucosal damage, usually with RTG as the active ingredient, and may also include pharmaceutically acceptable carriers or excipients; optional excipients include chitosan, cholesterol, liposomes or cyclodextrin; optional carriers include microspheres or microcapsules, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the comparison results of DAI scores of rats in each group;

[0013] Figure 2 This is a schematic diagram of the comparison results of MDI scores of rats in each group;

[0014] Figure 3 Schematic diagram of the comparison of TNF-α, IL-1β and IL-6 levels in the ileal mucosa of rats in each group;

[0015] Figure 4 Schematic diagram of immunohistochemical staining of ileum mucosa and expression detection results of D1R and D2R in rats of each group;

[0016] Figure 5 This is a schematic diagram of the comparison of TDI scores of the ileal mucosa of rats in each group. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained from commercial sources.

[0018] The experimental materials used in the following examples include:

[0019] 1. Experimental animals: 22 SPF male SD rats (provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected, with a body weight of 180-220 g, and the experimental animal license number was SCXK (Beijing) 2019-0510. The rats were kept in the Xinxiang Key Laboratory of Molecular Psychiatry, with free access to food, and maintained at an ambient temperature of 22-24 ° C, humidity <60%, and a 12-h light-dark cycle. The relevant regulations on experimental animal management and animal welfare were strictly followed during the feeding and experimental process.

[0020] 2. Main reagents:

[0021] TNBS (SigmaAldich, USA); rotigotine (Aladdin, China); rabbit anti-mouse D1 antibody (Alomone, Israel); rabbit anti-mouse D2 antibody (Alomone, Israel); SP-9002 rabbit immunohistochemistry detection kit (Zhongshan Jinqiao, China); DAB color development kit (Biyuntian, China); ELISA kit (for detection of TNF-α, IL-1β, and IL-6, respectively) (Shanghai ELISA Biotechnology Co., Ltd.).

[0022] Example 1 Experimental grouping and model construction

[0023] 1.1 Animal model production:

[0024] Trinitrobenzene sulfonic acid (TNBS) ileitis in rats was established as a Crohn's disease model according to the method described in "A novel diamino-pyridine derivative (IS-741) attenuates ratileitis induced by trinitrobenzene sulfonic acid". 5% (W / V) TNBS was mixed with 50% ethanol at a ratio of 1:1 to prepare enema solution. Rats were fed adaptively under standard laboratory conditions for 3 days and then fasted for 36 hours. After intraperitoneal injection of pentobarbital (40 mg / kg) for anesthesia, mid-abdominal laparotomy was performed, and TNBS solution (160 mg / kg, 50% ethanol) was injected into the ileum 10 cm near the ileocecal valve to induce rat ileitis.

[0025] 1.2 Grouping

[0026] According to the principle of completely randomized design, the rats were divided into a blank control group (Control, n = 6), a model group (Model, n = 8) and a treatment group (Model + RTG, n = 8). The rats in the model group and the treatment group were the model rats constructed in 1.1 above; the rats in the blank control group were infused with an equal volume of physiological saline solution. One day after modeling, the Model + RTG group was given an intraperitoneal injection of RTG (the dose was 10 mg / kg, and 0.2 ml of distilled water was added to prepare the solution), and the other groups were intraperitoneally injected with an equal amount of distilled water for 21 days;

[0027] 1.3 Statistical methods

[0028] GraphpadPrism 5.0 software was used for statistical analysis. The measurement data were expressed as mean ± standard error (mean ± SEM). The data that conformed to the normal distribution were analyzed by one-way analysis of variance (one-way ANOVA), and the Bonferroni test was used for the comparison between the two groups; the data that did not conform to the normal distribution were analyzed by the Kruskal-Wallis nonparametric test, and the k test was used for the comparison between the two groups. P < 0.05 was considered statistically significant.

[0029] Example 2 Experimental process and results

[0030] 2.1 General status observation and disease activity index (DAI) score

[0031] Experimental methods: The weight, coat color, diet, spirit, activity and stool of each group of rats were observed and recorded daily. DAI was scored according to the following criteria: weight loss (none: 0 points; 1%-5%: 1 point; 6%-10%: 2 points; 11%-15%: 3 points; >15%: 4 points); stool characteristics (normal: 0 points; semi-loose stool: 2 points; loose stool: 4 points); fecal occult blood (negative: 0 points; positive: 2 points; macroscopic bloody stool: 4 points). The DAI score is the sum of the above three indicators, and the score is scored once a week. The higher the score, the higher the disease activity.

[0032] Experimental results: The rats in the Control group were in good spirits, had shiny hair, were active, gradually gained weight, and had normal bowel movements. The rats in the Model group were listless, had dull hair, were slow to move, gained weight slowly, and had paste-like feces around the anus.

[0033] Compared with the Model group, the mental state, hair gloss, behavior and weight of rats in the Model+RTG group were improved. One rat in the Model group died during the experiment and was not included in the result analysis.

[0034] The DAI scoring results are as follows: Figure 1 As shown in the figure, compared with the Control group, the DAI scores of the rats in the Model group were significantly increased on the 7th, 14th and 21st days (P<0.001). Compared with the rats in the Model group, the DAI scores of the rats in the Model+RTG group showed a downward trend on the 7th and 14th days (P>0.05), and significantly decreased on the 21st day (P<0.01).

[0035] 2.2 Macroscopic damage index (MDI) score

[0036] Experimental methods: Rats were anesthetized and then killed. The ileum tissue of each group of rats was taken about 3 cm away from the ileocecal part, the intestinal cavity was washed with PBS, and the intestinal cavity was cut longitudinally along the mesentery. MDI was used for scoring, and the standards were: normal (0 points); local mucosal congestion and edema (1 point); linear ulcer (2 points); mucosal congestion and edema combined with linear ulcer (3 points); linear ulcers in more than 2 parts (4 points); longitudinal ulcer length > 1 cm (5 points); ulcer length > 2 cm (6 points).

[0037] Experimental results: Figure 2 As shown in the figure, compared with the Control group, the MDI score of the rats in the Model group was significantly increased (P<0.001). Compared with the rats in the Model group, the MDI score of the rats in the Model+RTG group was significantly decreased (P<0.05).

[0038] 2.3 Detection of pro-inflammatory factors in ileal mucosa

[0039] Experimental method: Take the ileum tissue of each group, cut it along the mesentery, rinse with PBS, fix it on the silicone plate with the mucosa facing up, and peel the mucosa layer with fine forceps and fine scissors under a stereomicroscope, wipe it dry with filter paper, and store it in a -80℃ refrigerator. Take about 10mg of tissue and put it into a 1.5ml EP tube, add 100μl of physiological saline, and prepare tissue homogenate. Protect from light, stand for 1h in an ice bath, centrifuge at 4℃ 12000rpm for 30min, and absorb the supernatant. According to the instructions of the ELISA kit (Shanghai ELISA Biotechnology Co., Ltd.), the levels of TNF-α, IL-1β and IL-6 in the tissues were detected respectively.

[0040] Experimental results: Figure 3 As shown in the results, compared with the Control group, the levels of TNF-α (P<0.001), IL-1β (P<0.001), and IL-6 (P<0.001) in the Model group were significantly increased. Compared with the Model group, the levels of TNF-α (P<0.05), IL-1β (P<0.01), and IL-6 (P<0.01) in the Model+RTG group were significantly decreased.

[0041] 2.4 Immunohistochemical staining to observe the histopathological changes of ileum tissue, the expression of D1R and D2R, and the tissue damage index (TDI)

[0042] Experimental method: The ileum tissues of rats in each group were fixed with 4% paraformaldehyde solution for 48 h, and sliced ​​(5 μm) using Leica CM1900 cryostat.

[0043] Immunohistochemical staining: incubate with 3% H2O2 for 10 min, wash with PBS for 5 min × 3 times, microwave antigen repair for 15 min, cool at room temperature for 30 min, block with goat serum for 30 min, add rabbit anti-rat D1R and D2R antibodies (1:100), and refer to the instructions of the immunohistochemical detection kit for the rest of the steps. After sealing with neutral gum, observe under an optical microscope.

[0044] TDI was scored according to the following criteria: normal (0 points); local mucosal and submucosal inflammatory infiltration and congestion and edema, mild mucosal erosion, and normal muscularis mucosa (1 point); based on a score of 1, the lesion range was >50% (2 points); obvious inflammatory infiltration and congestion and edema, ulcers extending to the submucosal layer and muscularis mucosa, and the muscularis mucosa was normal (3 points); based on a score of 3, the lesion range was >50% (4 points); significant and extensive ulcers, with cell necrosis reaching the muscularis mucosa (5 points); based on a score of 5, the lesion range was >50% (6 points).

[0045] Experimental results:

[0046] like Figure 4 As shown in the figure, the epithelial cells of the ileum mucosa of rats in the Control group were arranged neatly, and no congestion, edema or ulcers were observed. The ileum villi of rats in the Model group had varying degrees of shedding and necrosis, and congestion, edema and inflammatory cell infiltration deep into the muscular layer. The villi structure of the Model+RTG group was clearer, and mild congestion, edema and inflammatory cell infiltration were observed in all layers. This shows that RTG has a certain repair effect on ulcerative lesions of the ileum mucosa.

[0047] Immunohistochemical staining results showed that D1R positive cells were distributed in the epithelial layer and lamina propria, muscularis and myenteric plexus of the ileal mucosa of rats in each group. The distribution of D2R positive cells was similar to that of D1 positive cells.

[0048] like Figure 5 As shown in the figure, compared with the Control group, the TDI score of the rats in the Model group was significantly increased (P<0.001). Compared with the rats in the Model group, the TDI score of the rats in the Model+RTG group was significantly decreased (P<0.01).

[0049] The above results showed that Crohn's disease model rats exhibited varying degrees of diarrhea, decreased activity and weight loss; pathological examination showed necrosis and shedding of villi in the ileum wall, and congestion, edema and inflammatory cell infiltration could reach the muscular layer.

[0050] Chronic treatment with RTG can significantly improve disease activity scores, alleviate ileal histological damage, and inhibit intestinal mucosal inflammation, indicating that RTG can effectively inhibit intestinal mucosal damage, promote ulcer repair, inhibit the increase of proinflammatory factors, and significantly improve the symptoms of Crohn's disease. It is feasible to expand the drug use of RTG to the treatment of Crohn's disease.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of rotigotine in the preparation of a medicament for treating ileal mucosal damage, characterized in that: The ileal mucosal damage is damage caused by inflammation of the terminal ileum caused by Crohn's disease.

2. The use according to claim 1, characterized in that The treatment of ileal mucosal damage is to repair ileal mucosal ulcers.

3. The use according to claim 2, characterized in that The dosage of rotigotine is 10 mg / kg.

4. The use according to any one of claims 1 to 3, characterized in that: The active ingredient of the drug for treating ileal mucosal damage is rotigotine; and the drug also includes pharmaceutically acceptable excipients.

Images