Composition and application thereof in preparation of medicine for treating ulcerative colitis

Abstract

The invention belongs to the technical field of medicines, and provides a composition and application thereof in preparation of medicines for treating ulcerative colitis. The composition comprises mesalazine and a compound A, the amino acid sequence of the compound A is Lys-Glu-Pro-Val-Pro-Gln-Ala-Lys-Pro-Arg-Lys-Val-Ala-Ala-Gln, and the amino acid sequence of the compound A is shown in the description. The composition shows an obvious synergistic interaction effect when being used for preparing medicines for treating ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular, relates to a composition and an application thereof in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis is an inflammatory process limited to the colon mucosa and submucosa. The disease is mostly located in the sigmoid colon and rectum, and can also extend to the descending colon or even the entire colon. Its pathology is long and often recurrent. Patients with ulcerative colitis often present with abdominal pain and distress, constant urge to defecate, heaviness in the anus, and discomfort in defecation, which is called "tenesmus". Patients can complain of a feeling of falling, and no feeling of defecation or very little defecation after squatting when they want to defecate.

[0003] In recent years, the incidence of ulcerative colitis has shown a clear upward trend worldwide.

[0004] At present, drugs are mostly used to treat ulcerative colitis. Mesalazine, also known as 5-aminosalicylic acid, is an organic compound with a chemical formula of C7H7NO3. It is the active ingredient of SASP for the treatment of ulcerative colitis. It has a significant inhibitory effect on inflammation of the intestinal wall. Mesalazine can inhibit the synthesis of prostaglandins that cause inflammation and the formation of inflammatory mediators leukotrienes, thereby significantly inhibiting inflammation of the intestinal mucosa. Mesalazine can inhibit the synthesis of prostaglandins in a dose-dependent manner and reduce the release of PGE2 in the human colonic mucosa.

[0005] However, mesalazine has serious toxic side effects. Long-term use can not only cause stomach discomfort but also lead to dependence. The disease will recur and cannot be cured. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the first object of the present invention is to provide a composition, which, by combining the compound A of the present application with mesalazine, exhibits an obvious synergistic effect in the preparation of a drug for treating ulcerative colitis, and can enhance the effect of treating ulcerative colitis.

[0007] In view of the above-mentioned deficiencies in the prior art, the second object of the present invention is to provide an application of a composition in the preparation of a drug for treating ulcerative colitis.

[0008] In order to achieve the above object, the solution adopted by the present invention is:

[0009] A composition comprises mesalazine and compound A; the amino acid sequence of compound A is Lys-Glu-Pro-Val-Pro-Gln-Ala-Lys-Pro-Arg-Lys-Val-Ala-Ala-Gln.

[0010] An application of the above composition in preparing a medicine for treating ulcerative colitis.

[0011] Furthermore, in a preferred embodiment of the present invention, the dosage form of the drug includes: at least one of a solution, a tablet, a capsule, a granule, a suppository, and a sustained-release preparation for oral or parenteral administration.

[0012] Furthermore, in a preferred embodiment of the present invention, the administration method of the drug includes at least one of oral administration, inhalation, intravenous injection and enema.

[0013] The beneficial effects of a composition provided by the present invention and its application in preparing a drug for treating ulcerative colitis are:

[0014] The composition provided by the present invention, by combining the compound A of the present application with mesalazine, presents an obvious synergistic effect in the preparation of a drug for the treatment of ulcerative colitis. Whether it is enema intervention after skin sensitization of male SD rats by oxazolone solution, inducing lesions in their colon, and establishing a rat ulcerative colitis model that meets the clinical characteristics of the onset, or enema intervention of male SD rats by TNBS, a single enema, inducing lesions in their colon, and establishing a rat ulcerative colitis model that meets the clinical characteristics of the onset, the composition provided by the present application, in the various damage manifestations of the animal colon and the degree of histopathological lesion characteristics, shows a significant decrease reaction compared with the drug efficacy degree of mesalazine alone treatment group animals under the condition of monotherapy in the same period, and the drug efficacy superposition effect it shows is statistically different from the drug efficacy degree of mesalazine alone treatment group animals in the same period (P<0.05). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are representative pictures of colon tissue ulcer lesions of animals in each experimental group in Experimental Example 1 of the present invention;

[0016] Figure 2 These are representative pictures of colon tissue ulcer lesions in animals of each experimental group in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0018] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0019] Example 1

[0020] This embodiment provides a composition, comprising a mesalazine solution and a compound A solution in a dosage ratio of 5:0.5; wherein the concentration of the mesalazine solution is 8.57 mg / ml; the solute of the mesalazine solution is mesalazine, and the solvent of the mesalazine solution is 0.9% sodium chloride injection; the solute of the compound A solution is compound A, and the amino acid sequence of compound A is:

[0021] Lys-Glu-Pro-Val-Pro-Gln-Ala-Lys-Pro-Arg-Lys-Val-Ala-Ala-Gln, the concentration of the compound A solution is 0.857 mg / ml; the solvent of the compound A solution is sterile water for injection.

[0022] Experimental Example 1

[0023] Experimental animals: SD rats, SPF grade, 27, male, animal weight 245.55-290.97 g, animals 8-9 weeks old; Source: Beijing Huafukang Biotechnology Co., Ltd.; Production license number: SCXK (Beijing) 2019-0008; Experimental Animal Certificate Number: No.110322221100534773.

[0024] Experimental groups: This experiment set up three groups, namely, a model control group, a mesalazine treatment group, and a composition treatment group provided in Example 1, 9 rats in each group, for a total of 27 rats.

[0025] Animal modeling: (1) Modeling materials: gloves, sterile cotton swabs, cotton balls, medical caps and syringes of different specifications, etc.; (2) Modeling method: TNBS (trinitrobenzene sulfonic acid) enema method: Experimental animals that have passed the adaptation period test are selected as efficacy experimental animals. The model is constructed the next day after fasting treatment. The model construction method is TNBS ethanol solution enema. The TNBS induction dose is 30 mg / animal, single enema. Based on the body weight and DAI (disease activity index) score of the model animals on the fourth day (D3), they are randomly divided into 3 experimental groups, including a model control group, a mesalazine treatment group (the concentration of the mesalazine solution is 8.57 mg / ml, the solute of the mesalazine solution is mesalazine, and the solvent of the mesalazine solution is 0.9% sodium chloride injection), and a composition treatment group provided in Example 1. Drug treatment was started on the fifth day (D4). The treatment cycle was 14 days. During the experimental cycle, all experimental groups of animals were fed with normal drinking water and food.

[0026] Dosing information is shown in Table 1:

[0027] Table 1

[0028]

[0029] Dosing volume: 3.5 ml / kg;

[0030] Administration site: Enema, 8-9 cm from the anus;

[0031] Date of administration: D4-D17;

[0032] Dosing time: During the treatment cycle, fasting begins at 5 a.m. the day before dosing, and dosing is completed at 10-11 a.m. on the day of dosing. Feeding is performed after dosing.

[0033] Fasting treatment at the end of the experiment: fasting began at 5:00 pm the day before the gross autopsy and continued until 1:30 pm on the day of the autopsy when the end point autopsy began;

[0034] The day of model induction was defined as the first day of the experiment (day 0, D0).

[0035] 1.1 Effects on body weight changes in model animals

[0036] Body weight measurement:

[0037] Test animals: All surviving experimental animals planned for testing;

[0038] Measurement time: The experimental animals were weighed twice during the adaptation period to evaluate the animal's adaptation status; they were weighed every day during the experimental period to evaluate the disease progression of the model animals; all animals were weighed at the end of the experiment to calculate the dosage of sodium pentobarbital.

[0039] The results of body weight measurement during the experimental period are shown in Table 2:

[0040] Table 2 Effects of each drug group on the weight gain of model animals

[0041] Group Treatment group (D3, g) Experimental endpoint (D17, g) The day of autopsy (D18, g) Weight gain (D17-D3,%) Model control group 245.54±29.87 279.77±26.20 264.53±26.62 14.49±7.52 Mesalazine treatment group 247.93±30.80 293.95±30.76 276.35±30.76 19.30±12.46 Combination treatment group 244.39±33.97 303.75±25.78 287.26±25.78 25.63±13.69*

[0042] Note: All data were statistically analyzed using mean ± standard deviation (SD), and “*” indicates the statistical analysis results of different treatment groups and the model control group.

[0043] The results in Table 2 show that the experimental animals showed a significant weight loss response after intervention with the model inducer TNBS. The mean body weight decreased from 265.61±9.81g on the day of model induction (D0) to 245.89±27.61g on the day before grouping treatment (D3). It dropped to the lowest value of 237.11±24.37g on the fifth day of the experiment (D4), and then began to recover. At the end of the experiment (D17), the mean body weight of the animals in the model control group was 279.77±26.20g, an increase of 34.22±15.24g compared with the day of grouping treatment. The weight gain during the treatment period was 14.49±7.52%.

[0044] The positive control drug mesalazine also had a promoting effect on the weight gain of the model animals under the set treatment plan. The mean body weight increased from 247.93±30.80g on the day of grouping to 293.95±30.76g at the end of the experiment, with a weight increase of 19.30±12.46%. There was no statistical difference compared with the model control group in the same period (P>0.05), and the weight gain promoting effect was poorer than that of the combination treatment group.

[0045] The combination treatment group had a certain promoting effect on the weight gain of the model animals. The mean body weight increased from 244.39±33.97g on the day of group treatment (D4) to 303.75±25.78g at the end of the experiment. The body weight increase during the experimental treatment period was 25.63±13.69%, which was statistically different from the model control group (P<0.05).

[0046] 1.2 Effect on the progression of disease activity in ulcerative colitis model animals

[0047] Determination of Disease Activity Index (DAI):

[0048] Test animals: Animals in each experimental group;

[0049] Determination method: The disease activity index of each group of experimental animals was scored based on animal weight, feces morphology and characteristics, occult blood or blood in stool. The scoring criteria are shown in Table 3;

[0050] Table 3 Scoring criteria for disease activity index of experimental animals in each drug administration group

[0051] Points Percentage of weight loss Stool consistency Fecal occult blood 0 normal normal Negative 1 1-5％ Semi-formed stool Occult blood weak positive 2 5-10％ Pasty stool Occult blood positive 3 10-20％ Loose stools Weak positive bloody stool 4 >20％ Watery stool Strong positive blood in stool

[0052] Note: DAI score is calculated based on the extent of weight loss, stool morphology and the progression of blood in stool, and the sum of the three scoring indicators is used for statistical analysis.

[0053] Measurement time: once a day for 14 consecutive days after model induction.

[0054] The DAI score results during the experimental period are shown in Table 4:

[0055] Table 4 DAI score results of animals in each experimental group during the test period

[0056] Group Treatment group (D3, points) Experimental day 10 (D10, minutes) Experimental endpoint (D17, min) AUC(D17-D3) Model control group 5.56±1.59 1.33±1.41 1.00±1.12 38.30±4.28 Mesalazine treatment group 5.67±1.87 1.00±1.80 0.33±1.00 28.11±4.19*** Combination treatment group 5.56±1.88 0.89±1.83 0.33±0.71 27.50±3.83****

[0057] Note: All data were statistically analyzed using mean ± standard deviation (SD), and “*” indicates the statistical analysis results of different treatment groups and the model control group.

[0058] The results in Table 4 show that the animals in the model control group showed obvious colitis disease characteristics after TNBS intervention, which were manifested by significantly inhibited weight gain, decreased animal activity level, pasty and thin feces, and inflammation and swelling around the anus. By the end of the experiment (D17), the disease manifestations basically returned to normal levels, with an average DAI score of 1.00±1.12 points. The AUC of the DAI score during the experimental period was (D17-D3) It is 38.30±4.28.

[0059] The positive control drug has a significant therapeutic effect on the disease progression of ulcerative colitis in model animals under the set treatment regimen, and its corresponding DAI score AUC (D17-D3) It was 28.11±4.19, which was statistically different from the model control group (P<0.05).

[0060] The combination treatment group had a significant therapeutic effect on the disease activity of ulcerative colitis in the model animals under the set treatment plan. Compared with the animals in the model control group during the same period, the weight loss and stool characteristics of the animals in the combination treatment group were significantly alleviated, and the mean DAI score decreased significantly. On the 10th day (D10) of the experiment, it began to recover to the level of normal animals, which was 0.89±1.83 points. The AUC of its DAI score during the experimental period was (D17-D3) It was 27.50±3.83, which was statistically different from the model control group (P<0.05).

[0061] 1.3 Effects on the pathological characteristics of colonic tissue in model animals

[0062] 1.3.1 Gross anatomy:

[0063] Dissected animals: All surviving experimental animals planned for measurement;

[0064] Dissection time: end point of the experiment (D18);

[0065] Anatomical method: The experimental animals were euthanized by sodium pentobarbital anesthesia and then subjected to gross dissection, anatomical observation and colon gross injury scoring. The descending colon segment (end of the cecum to the anus) tissue was obtained, and the intestinal segment was washed with saline and then weighed and measured in length. Intestinal segment image data was collected, and the colon ulcer surface was photographed under a stereo microscope to calculate the ulcer area.

[0066] 1.3.2 Colon histopathological examination:

[0067] Tissue sampling: All colon tissues of the surviving experimental animals planned for measurement (2 cm from the anus to the cecum) were divided into upper section (close to the cecum), middle section and lower section (close to the anus) by cross-section;

[0068] Tissue processing: fixed with 10% formaldehyde, embedded in paraffin, cut into 3 μm thick sections, then dewaxed in xylene, hydrated with graded ethanol, stained with H&E, dehydrated, transparent, and sealed, and the pathological changes of colon tissue were observed and photographed under a microscope;

[0069] Pathological diagnosis and analysis: Based on the results of colon H&E staining, the colon inflammation and colon ulcer of animals in each experimental group were analyzed. The evaluation indicators are shown in Table 5;

[0070] Detection time: Pathological specimens were prepared 48 hours after routine fixation.

[0071] Table 5 Scoring criteria for colon histological injury

[0072]

[0073] (1) Effects on the gross morphological damage characteristics of the colon in model animals

[0074] In this experiment, the animals in each experimental group were euthanized and the colon was grossly dissected on the day after the last administration. The gross dissection revealed that the colon segments of the animals in the model control group showed obvious atrophy and deformation, manifested by significant thickening of the intestinal wall, bleeding and ulcers, etc. The ulcers were multifocal and adhered to the surrounding tissues, which were most serious in the middle and terminal segments of the colon. At the same time, obvious segmental lesions appeared in the middle and lower segments of the colon, the intestine was narrow at the lesion site, and fecal congestion formed swelling, resulting in a significant enlargement of the intestinal longitudinal axis. When the intestine was dissected and laid flat on filter paper, obvious ulcer lesions were seen, showing intestinal tissue erosion and bleeding.

[0075] Table 6 Effects of each group on colon ulcer in model animals

[0076] Group <![CDATA[Colonic ulcer area (cm 2 )]]> Model control group 3.197±2.180 Mesalazine treatment group <![CDATA[0.530±0.353 ** ]]> Combination treatment group <![CDATA[0.424±0.373 ** ]]>

[0077] Note: Colon tissues of animals in each experimental group were collected at the end of the experiment to calculate the ulcer area. All individual data were statistically analyzed using mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of different experimental groups and the model control group.

[0078] The results of collecting ulcer images and calculating the ulcer area using a stereomicroscope (Table 6) showed that under the experimental conditions, the animals in the model control group developed obvious colon ulcerative lesions after TNBS induction, with an area of ​​3.197±2.180cm 2 .

[0079] The results in Table 6 show that the positive control drug mesalazine has a significant therapeutic effect on the colon ulcer damage of the model animals under the set treatment regimen. Compared with the model control group animals, the colon ulcer area of ​​the treatment group animals was significantly reduced to 0.530±0.353cm 2 , with statistically significant difference (P<0.05). At the same time, the degree of intestinal adhesion and segmental lesions showed obvious improvement.

[0080] The combination treatment group showed a certain degree of ulcer repair superposition effect under the set treatment scheme. The area of ​​colon ulcer in the corresponding treatment group animals showed a certain degree of decrease compared with the positive control drug treatment condition, which was 0.424±0.373cm 2 .

[0081] (2) Effect on colon weight / length ratio of model animals

[0082] Figure 1 The following are representative images of colon histopathology of animals in each experimental group. The colon was diagnosed and analyzed under 100× and 200× optical microscopes. Three animals in each experiment were selected for the results display. The image scales were 1 mm and 500 μm, respectively. Table 7 shows the effects of each experimental group on the atrophy process of the model animals:

[0083] Table 7 Effects of each group on the atrophy process of model animals

[0084] Group Colon weight (g) Colon length (cm) Colon weight / length (100*g / cm) Model control group 2.345±0.701 13.83±1.98 17.33±5.54 Mesalazine treatment group <![CDATA[1.600±0.188 ** ]]> 15.36±1.29 <![CDATA[10.50±1.60 ** ]]> Combination treatment group <![CDATA[1.535±0.139 ** ]]> <![CDATA[18.11±1.41 **** ]]> <![CDATA[8.54±1.21 ***## ]]>

[0085] Note: At the end of the experiment, the colon tissues of the animals in each experimental group were collected, the length and weight were measured, and the colon / weight ratio and ulcer area were calculated. All individual data were statistically analyzed using the mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of different experimental groups and model control groups. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 represents the statistical analysis result of the mesalazine treatment group and the combination treatment group.

[0086] The results of calculating the length and weight of the colon segments in each experimental group showed that ( Figure 1 and Table 7), after TNBS induction, the colon tissue of the model animals showed obvious atrophy, swelling and deformation, and the colon weight / length ratio showed a significant increase, which was 17.33±5.54 100*g / cm.

[0087] The positive control drug mesalazine had a significant therapeutic effect on the atrophy and swelling deformation process of the model animals under the set treatment plan. The colon weight / length ratio was 10.50±1.60 100*g / cm, and the degree of improvement was statistically significant compared with the model control group (P<0.05).

[0088] Under the treatment of the combination treatment group, compound A was able to promote the therapeutic effect of mesalazine to a certain extent. The colon weight / length ratio of the animals in the corresponding treatment group showed a certain degree of decrease compared with that under the single drug treatment condition, which was 8.54±1.21 100*g / cm, and its pharmacodynamic additive effect was statistically different from the pharmacodynamic degree of the animals in the mesalazine single treatment group during the same period (P<0.05).

[0089] (3) Effects on colon tissue ulcers and inflammatory response scores in model animals

[0090] Table 8 shows the colon histopathological diagnosis results at the experimental endpoint:

[0091] Table 8 Effects of each experimental group on the characteristics of colon histopathological lesions in model animals

[0092]

[0093] Note: Based on H&E staining of the colon of each experimental group of animals, the colon ulcer and inflammatory infiltration of each experimental group of animals were diagnosed, analyzed and scored according to the "Colonic Histological Injury Scoring Standard". All individual data were statistically analyzed using the mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of different experimental groups and model control groups. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 represents the statistical analysis result of the mesalazine treatment group and the combination treatment group.

[0094] The results in Table 8 show that in this experimental system, the animals in the model control group showed obvious histological changes of colitis, especially the pathological changes in the middle part of the colon. Therefore, pathological diagnosis and analysis were performed on the middle part of the colon. The histopathological lesions were mainly characterized by significant thickening of the colon wall, formation of fissure-like ulcers on the surface of the intestinal mucosa, typical granulomas in the submucosal layer and muscular layer, accompanied by inflammatory cell infiltration mainly composed of lymphocytes and plasma cells, small vessel arterial endarteritis and fibroblast activation, thickening of the arteriolar wall and stenosis of the lumen, and obvious connective tissue hyperplasia and fibrosis in the submucosal layer and muscular layer. The fibers were distributed in bundles, and the destruction of the crypt structure and bleeding were observed around the ulcer. The mean histopathological scores of tissue ulcer and inflammatory reaction were 1.78±0.44 and 1.89±0.33, respectively, and the mean pathological scores of colon fibrosis and goblet cell damage were 1.33±0.71 and 0.44±0.53, respectively.

[0095] The positive control drug mesalazine had a significant inhibitory effect on the colon ulcer and inflammatory response process of the model animals under the set treatment plan. Compared with the animals in the model control group, the degree of colon tissue ulcer and inflammation in the corresponding treatment group animals were significantly alleviated, and the mean pathological scores were 0.56±0.73 points and 1.00±0.00 points, respectively. The degree of improvement was statistically different from that of the model control group in the same period (P<0.05).

[0096] At the same time, the connective tissue hyperplasia and fibrosis process of the colon submucosal and muscularis of the animals in the positive drug treatment group showed significant improvement and healing. Cell hyperplasia was observed in both the submucosal and muscularis layers. The proliferating cells were irregularly arranged, and a small amount of fibers were distributed in the intercellular spaces of the muscularis layer. The degree of fibrosis and goblet cell injury scores were 0.33±0.50 and 0, respectively, which were statistically different from those of the model control group in the same period (P<0.05).

[0097] In the combination treatment group, under the set treatment regimen, compound A promoted the therapeutic effect of mesalazine to a certain extent. The colon ulcer manifestations, inflammatory infiltration and intestinal fibrosis degrees of the corresponding treatment group animals showed a certain degree of decrease compared with those under single-drug treatment conditions, which were 0.22±0.67 points, 1.00±0.00 points and 0.11±0.33 points, respectively, which were statistically different from the model control group in the same period (P<0.05).

[0098] In summary, this experiment established a rat ulcerative colitis model that is consistent with the clinical characteristics of the disease. The results of the efficacy evaluation in this model showed that the combination treatment group had a significant therapeutic effect, and the combination of compound A and mesalazine under the above conditions had a certain synergistic effect on the treatment of ulcerative colitis.

[0099] Experimental Example 2

[0100] Experimental animals: SD rats, SPF grade, 40, male, animal weight 261.12-297.36g, animals 8-9 weeks old; Source: Beijing Huafukang Biotechnology Co., Ltd.; Production license number: SCXK (Beijing) 2019-0008; Animal certificate number: NO.110322221101529346.

[0101] Experimental groups: This experiment set up 4 groups, namely normal control group, model control group, mesalazine treatment group, and combination treatment group, 10 rats in each group, for a total of 40 rats.

[0102] Animal modeling:

[0103] Oxazolone presensitization + enema two-step method: The experimental animals that passed the adaptation period test were selected as the efficacy experimental animals, and were randomly divided into a normal control group and a model induction group according to their body weight. Except for the animals in the normal control group who were intervened by solvent, the other animals in the model induction group were intervened by oxazolone to induce ulcerative colitis model. The model construction method is as follows:

[0104] (1) Model pre-sensitization stage: On the day of grouping (D1), 3% oxazolone solution was applied to the abdominal skin of the experimental animals (depilated), and repeated every other day, with an application volume of 200 μL / animal, for 2 consecutive times;

[0105] (2) Enema induction stage: On the 7th day (D7) of the experiment, the animals in the model induction group were sub-grouped according to the body weight parameters and divided into three experimental groups, namely, a model control group, a mesalazine treatment group (the concentration of the mesalazine solution was 8.57 mg / ml, the solute of the mesalazine solution was mesalazine, and the solvent of the mesalazine solution was 0.9% sodium chloride injection), and a composition treatment group provided in Example 1. Enema treatment was performed with 1% oxazolone solution, a single enema, and drug treatment was started on the next day (D8) after grouping. The cycle was 14 days, during which all animals were routinely fed.

[0106] See Table 9 for dosing information:

[0107] Table 9

[0108]

[0109] Dosing volume: 3.5 ml / kg;

[0110] Administration site: Enema administration via the anus;

[0111] Dosing date: D8-D21, a total of 14 days;

[0112] Fasting treatment at the end of the experiment: After fasting for 16 hours at the end of the experiment, gross anatomical specimens were obtained;

[0113] The day of model induction was defined as the first day of the experiment (day 1, D1).

[0114] 2.1 Effects on body weight changes in model animals

[0115] Body weight measurement:

[0116] Test animals: All surviving experimental animals planned for testing;

[0117] Measurement time: The experimental animals were weighed twice during the adaptation period to evaluate the animal's adaptation status; they were weighed every day during the experimental period to evaluate the disease progression of the model animals; all animals were weighed at the end of the experiment to calculate the dosage of sodium pentobarbital and the relevant organ-body ratios of the animals.

[0118] The results of body weight measurement during the experimental period are shown in Table 10.

[0119] Table 10 Effects of each drug administration group on the weight gain of model animals

[0120]

[0121] Note: All data were statistically analyzed using mean ± standard deviation. “*” indicates the statistical analysis results compared with the normal control group; “#” indicates the statistical analysis results compared with the model control group.

[0122] The results in Table 10 show that oxazolone skin sensitization had no significant effect on the weight gain of experimental animals. The weight of animals in the model induction group showed normal growth, from 280.78±7.67g on the day of grouping (D1) to 298.61±13.41g on the sixth day of the experiment (D6), which was consistent with the weight gain of animals in the normal control group during the same period (279.06±8.57g and 299.93±10.12g), and no difference was observed.

[0123] The weight of animals in the normal control group increased steadily, with the mean weight increasing from 284.19±8.57g on the day of grouping in the second stage (D7) to 299.12±16.29g at the end of the experiment (D21). The weight increase during the experimental period was 5.20±3.29%.

[0124] The mean body weight of the model control animals at the end of the experiment (D21) was 284.52±14.80g, which was only 3.76±12.81g higher than that on the day of group treatment (280.76±8.16g). The body weight increase during the experimental period was 1.35±4.63%, which was statistically different from the normal control group during the same period (P<0.05).

[0125] The positive control drug mesalazine had a certain promoting effect on the weight gain of the model animals under the set treatment plan. The mean body weight increased from 282.72±10.06g on the day of treatment grouping (D7) to 292.05±16.64g at the experimental endpoint. The weight gain during the experimental treatment period was 3.32±5.06%, but there was no statistical difference in its weight gain promoting effect compared with the model control group during the same period (P>0.05).

[0126] The mean body weight of the animals in the combination treatment group increased from 281.41±9.02g on the day of grouping to 295.94±13.37g at the end of the experiment, with a body weight increase of 5.16±3.21%. The degree of weight gain promotion was significantly different from that of the model control group during the same period (P<0.05). The results of this experiment suggest that compound A has a certain sensitizing effect on the symptom improvement effect of mesalazine in the treatment of ulcerative colitis.

[0127] 2.2 Effect on the progression of disease activity in ulcerative colitis model animals

[0128] Determination of Disease Activity Index (DAI):

[0129] Test animals: Animals in each experimental group;

[0130] Determination method: The disease activity index of each group of experimental animals was scored based on animal weight, feces morphology and characteristics, occult blood or blood in stool. The scoring criteria are shown in Table 11;

[0131] Table 11 Scoring criteria for disease activity index of experimental animals in each group

[0132]

[0133] Note: DAI score is calculated based on the extent of weight loss, stool morphology and the progression of blood in stool, and the sum of the three scoring indicators is used for statistical analysis.

[0134] Measurement time: 8th day after model induction, once a day for 14 consecutive days.

[0135] The DAI score results during the experimental period are shown in Table 12:

[0136] Table 12 DAI score results of animals in each experimental group during the test period

[0137] Group Experimental day 9 (D9, minutes) Experimental day 16 (D16, points) Experimental endpoint (D21, min) AUC(D7-D21) Normal control group 0.70±0.67 0.20±0.42 0.00±0.00 4.60±1.17 Model control group <![CDATA[4.80±0.92 **** ]]> <![CDATA[2.30±1.49 **** ]]> <![CDATA[1.00±0.94 **** ]]> <![CDATA[37.90±2.70 **** ]]> Mesalazine treatment group 3.40±1.71 1.50±1.51 0.50±0.85 <![CDATA[24.65±3.79 ### ]]> Combination treatment group <![CDATA[2.80±1.81 # ]]> <![CDATA[0.60±0.52 ## ]]> <![CDATA[0.00±0.00 ## ]]> <![CDATA[14.30±2.73 ###+++ ]]>

[0138] Note: All data were statistically analyzed using mean ± standard deviation (SD). * P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001 indicates the statistical analysis results of the model control group and the normal control group. # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001 indicates the statistical analysis results between animals in different treatment groups and the model control group; + P<0.05, ++ P<0.01, +++ P<0.001, ++++ P<0.0001 represents the statistical analysis result between the mesalazine treatment group and the combination treatment group.

[0139] The results in Table 12 show that compared with the animals in the normal control group, the animals in the model control group developed typical symptoms of ulcerative colitis after oxazolidinone enema, which were specifically manifested as suppressed weight gain, perianal inflammation and swelling, pasty and thin feces, and decreased animal activity levels, but no obvious bleeding or occult blood was observed (all tests were negative). The disease symptoms were most obvious 2-3 days after model induction (D9 and D10), and the corresponding DAI score average was 4.80±0.92 points. Subsequently, the disease symptoms began to subside, and the score gradually decreased. At the end of the experiment (D21), the weight loss performance basically returned to normal, and the fecal characteristics showed loose stools, and the corresponding DAI score average was 1.00±0.94 points. The area under the curve of the DAI score during the experimental period AUC (D7-D21) The mean value was 37.90±2.70, which was statistically significant compared with the normal control group (4.60±1.17) (P<0.05).

[0140] The positive control drug mesalazine had a significant therapeutic effect on the disease activity of ulcerative colitis in the model animals under the set treatment plan. Compared with the model control group animals in the same period, the weight loss and stool characteristics of the animals in the mesalazine treatment group were significantly alleviated, and the mean DAI score decreased significantly. On the 9th day of the experiment (D9), the mean DAI score was 3.40±1.71 points, and the AUC of the DAI score during the experimental period was (D7-D21) It was 24.65±3.79, which was statistically different from the model control group (P<0.05).

[0141] In the combination treatment group, under the set treatment conditions, compound A had a significant sensitizing effect on the therapeutic effect of the positive control drug mesalazine, and its corresponding DAI score AUC (D7-D21) It was 14.30±2.73, which was significantly lower than that of the mesalazine monotherapy group, and the difference was statistically significant (P<0.05).

[0142] 2.3 Effects on the pathological characteristics of colonic tissue in model animals

[0143] 2.3.1 Gross anatomy:

[0144] Dissected animals: All surviving experimental animals planned for measurement;

[0145] Dissection time: end point of the experiment (D22);

[0146] Dissection method: The experimental animals were euthanized by sodium pentobarbital anesthesia and then subjected to gross dissection, anatomical observation and colon gross injury scoring (see Table 13 for scoring criteria). The descending colon tissue was obtained, and the intestinal segment was washed with saline before being weighed and measured. Intestinal segment image data was collected, and the colon ulcer surface was photographed under a stereo microscope to calculate the ulcer area.

[0147] 2.3.2 Colonic histopathological examination:

[0148] Tissue sampling: All colon tissues of the surviving experimental animals planned for measurement (2 cm from the anus to the cecum) were divided into upper section (close to the cecum), middle section and lower section (close to the anus) by cross-section;

[0149] Tissue processing: fixed with 10% formaldehyde, embedded in paraffin, cut into 3 μm thick sections, then dewaxed in xylene, hydrated with graded ethanol, stained with H&E, dehydrated, transparent, and sealed, and the pathological changes of colon tissue were observed and photographed under a microscope;

[0150] Pathological diagnosis and analysis: Based on the results of colon H&E staining, the colon inflammation and colon ulcer of the animals in each experimental group were analyzed (the scoring criteria are shown in Table 13);

[0151] Detection time: Pathology films are completed within one week of sample collection, and pathology diagnosis is analyzed within one month after the completion of the pathology films.

[0152] Table 13 Scoring criteria for colon histological injury

[0153]

[0154]

[0155] (1) Effects on the gross morphological damage characteristics of the colon in model animals

[0156] In this experiment, the animals in each experimental group were euthanized and the colon was grossly dissected the day after the last administration. The gross dissection revealed that compared with the normal control group, the colon segments of the animals in the model control group showed obvious atrophy, deformation and tissue ulceration, and the intestinal wall showed obvious thickening and intestinal adhesion, with the most obvious lesions in the middle and lower segments of the colon; obvious segmental lesions appeared in the middle and lower segments of the colon, the lesion site was narrow, and the feces accumulated to form swelling, resulting in a significant enlargement of the longitudinal axis of the intestine. When the intestine was longitudinally cut open and laid flat on filter paper, multiple ulcer erosion foci on the inner wall of the colorectum were visible, showing a fusion distribution and bleeding.

[0157] Table 14 Effects of each group on colon ulcer in model animals

[0158] Group <![CDATA[Colonic ulcer area (cm 2 )]]> Normal control group 0.000 Model control group <![CDATA[2.743±1.181 **** ]]> Mesalazine treatment group <![CDATA[0.520±0.741 #### ]]> Combination treatment group <![CDATA[0.166±0.314 #### ]]>

[0159] Note: Colon tissues of animals in each experimental group were collected at the end of the experiment to calculate the ulcer area. All individual data were statistically analyzed using mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of the model control group and the normal control group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 indicates the statistical analysis results of animals in different treatment groups and the model control group.

[0160] The results of using a stereo microscope to collect ulcer images and calculate the ulcer area (Table 14) showed that under the experimental conditions, the colon of the animals in the model control group showed obvious ulcer lesions after oxazolidinone induction, with an area of ​​2.743±1.181cm 2 .

[0161] The results in Table 14 show that the positive control drug mesalazine has a significant therapeutic effect on colon ulcer damage in model animals under the set treatment regimen. Compared with the model control group animals, the colon ulcer area of ​​the treatment group animals was significantly reduced to 0.520±0.741cm 2 The degree of improvement was statistically significant compared with the model control group (P<0.05). At the same time, the symptoms of intestinal adhesion and segmental lesions were significantly improved.

[0162] In the combined treatment group, compound A had a significant sensitizing effect on the therapeutic repair effect of mesalazine under the set treatment regimen. The area of ​​colon ulcer in the corresponding treatment group was significantly reduced compared with that under the single drug treatment condition, which was 0.166±0.314cm 2The drug-effectiveness additive effect was statistically different from that of the animals in the mesalazine monotherapy group during the same period (P<0.05).

[0163] (2) Effect on colon weight / length ratio of model animals

[0164] Figure 2 The following are representative images of colon histopathology of animals in each experimental group. The colon was diagnosed and analyzed under 100× and 200× optical microscopes. Three animals were selected from each experimental group to display the results. The image scales were 1 mm and 500 μm, respectively. Table 15 shows the effects of each experimental group on the atrophy process of the model animals:

[0165] Table 15 Effects of each group on colon ulcer in model animals

[0166] Group Colon weight (g) Colon length (cm) Colon weight / length (100*g / cm) Normal control group 1.93±0.18 19.84±2.04 9.74±0.31 Model control group <![CDATA[2.49±0.48 ** ]]> <![CDATA[16.95±0.90 *** ]]> <![CDATA[14.67±2.57 **** ]]> Mesalazine treatment group 2.20±0.19 <![CDATA[21.77±0.83 ### ]]> <![CDATA[10.13±0.87 ### ]]> Combination treatment group <![CDATA[2.00±0.11 ###++ ]]> <![CDATA[22.02±1.36 ### ]]> <![CDATA[9.10±0.40 ###++ ]]>

[0167] Note: At the end of the experiment, the colon tissue of each experimental group was collected, the length and weight were measured, and the colon / weight ratio was calculated. All individual data were statistically analyzed using the mean (Mean) ± standard deviation (SD). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of the model control group and the normal control group. # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001 indicates the statistical analysis results between animals in different treatment groups and the model control group; + P<0.05, ++ P<0.01, +++ P<0.001, ++++ P<0.0001 indicates the statistical analysis results between the mesalazine treatment group and the combination treatment group.

[0168] The results of calculating the length and weight of the colon segments in each experimental group showed that ( Figure 2 The colon tissue of the model animals induced by oxazolidinone showed obvious atrophy, swelling and deformation, and the colon weight / length ratio increased to 14.67±2.57100*g / cm, which was statistically different from the level of the normal control group animals (9.74±0.31 100*g / cm) (P<0.05).

[0169] The positive control drug mesalazine had a significant therapeutic effect on the atrophy and swelling deformation process of the model animals under the set treatment plan. The colon weight / length ratio was significantly decreased to 10.13±0.87 100*g / cm, which was statistically different from the model control group (P<0.05).

[0170] The combination treatment group significantly improved the colon damage of the model animals, and the colon weight / length ratio was 9.10±0.40 100*g / cm, which was significantly different from the model control group (P<0.001). At the same time, the combination treatment showed a significant synergistic effect on the effect of mesalazine, and the colon weight / length ratio of the animals in this group was significantly lower than that of the mesalazine monotherapy group (P<0.01).

[0171] (3) Effects on colon tissue ulcers and inflammatory response scores in model animals

[0172] Table 16 shows the colon histopathological diagnosis results at the experimental endpoint:

[0173] Table 16 Effects of each experimental group on the characteristics of colon histopathological lesions in model animals

[0174]

[0175] Note: Based on H&E staining of the colon of the animals in each experimental group, the histopathological diagnosis, analysis and scoring of colon mucosal epithelial injury and ulcer formation, lymphocyte mononuclear and plasma cell inflammatory infiltration and crypt structure destruction of the animals in each experimental group were performed according to the "Colonic Histological Injury Scoring Standard". All individual data were statistically analyzed using the mean (Mean) ± standard deviation (SD). * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 indicates the statistical analysis results of the model control group and the normal control group. # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001 indicates the statistical analysis results between animals in different treatment groups and the model control group; + P<0.05, ++ P<0.01, +++ P<0.001, ++++ P<0.0001 represents the statistical analysis result between the mesalazine treatment group and the combination treatment group.

[0176] The results in Table 16 show that in this experimental system, the colorectal tissue structure of the animals in the normal control group was normal, with clear stratification, innate immune cell infiltration was observed in the colon propria, and no obvious histological abnormalities were observed in the rest. The animals in the model control group showed obvious histological characteristic lesions of colitis under the induction of oxazolone, mainly including ulcers formed by mucosal epithelial necrosis, typical granulomas in the submucosal layer and muscular layer, accompanied by inflammatory cell infiltration mainly composed of lymphocytes and plasma cells, and abnormal and destroyed crypt structures, etc. The depth of the lesions could reach the muscular layer, and some animals could reach the serosa layer. The changes in the crypt structure were mainly dilation and swelling, and crypt abscesses were observed in the local lesion area; at the same time, inflammation and structural destruction of arterioles (thickening or stenosis of the wall, accompanied by fibrinoid necrosis) and fibroblast activation were observed in the granuloma tissue. Based on the characteristics of colonic histological lesions in the model control group, the colonic epithelial injury and ulceration, inflammatory infiltration of lymphocytes and mononuclear plasma cells, crypt abscesses and structural destruction in the experimental groups were scored for histopathological diagnosis (including the degree and depth of lesions) using the "Colonic Histological Injury Scoring Standard". The mean scores were 5.70±0.48, 5.20±0.92 and 2.90±0.32, respectively. The pathological diagnosis and scoring results suggest that the progression of ulcerative colitis in the model control group under this experimental system is in the stage of acute to chronic inflammation, which is consistent with the symptoms of clinical ulcerative colitis from active stage to remission stage.

[0177] The positive control drug mesalazine had a significant inhibitory effect on the colon ulcer, inflammatory response and crypt destruction process of the model animals under the set treatment scheme. Compared with the animals in the model control group, the colon mucosal epithelial ulcer, inflammatory infiltration and crypt destruction of the corresponding treatment group animals were in a repaired state. The mean mucosal epithelial ulcer pathological score was 2.00±0.00 points, the mean chronic inflammatory (lymph, monocyte and plasma cell) infiltration score was 2.70±0.48 points, and the mean crypt injury score was 1.00±0.00 points. The degree of improvement in different colon injury characteristics was statistically significant compared with the model control group in the same period (P<0.05).

[0178] Under the set treatment regimen, compound A had a significant synergistic effect on the therapeutic effect of mesalazine in the combination treatment group. The corresponding combined treatment group animals showed better therapeutic performance in colon ulcers, inflammatory responses and crypt structure damage than those treated with mesalazine alone. The corresponding mean pathological scores were 1.40±0.97 points, 2.30±0.95 points and 0.80±0.42 points, respectively, which were significantly lower than the therapeutic effects under mesalazine monotherapy.

[0179] In summary, this experiment established a rat ulcerative colitis model that is consistent with the clinical characteristics of the disease. The results of the efficacy evaluation in this model showed that the combination treatment group had a significant therapeutic effect, and the combination of compound A and mesalazine under the above conditions had a certain synergistic effect on the treatment of ulcerative colitis.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition, characterized in that Comprising mesalazine and compound A; the amino acid sequence of compound A is: Lys-Glu-Pro-Val-Pro-Gln-Ala-Lys-Pro-Arg-Lys-Val-Ala-Ala-Gln.

2. Use of the composition as claimed in claim 1 in the preparation of a drug for treating ulcerative colitis.

3. The use according to claim 2, characterized in that: The dosage form of the drug includes: solution, tablet, capsule, granule, suppository and at least one of oral or parenteral sustained-release preparations.

4. The use according to claim 2, characterized in that: The administration method of the drug includes at least one of oral administration, inhalation, intravenous injection and enema.

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