Use of recombinant apolipoprotein j in the preparation of a medicament for treating ulcerative colitis

Abstract

The application discloses a new use of recombinant apolipoprotein J in preparation of a medicine for treating ulcerative colitis, and belongs to the field of biological medicines. The DSS-induced ulcerative colitis model animal experiment shows that the recombinant apolipoprotein J has certain curative effect on the body weight loss, diarrhea, hematochezia and intestinal injury caused by ulcerative colitis, and provides a new medicine selection for the treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of recombinant apolipoprotein J in the preparation of medicaments for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a refractory disease characterized by chronic nonspecific inflammation of the intestines. It is one of the most serious gastrointestinal diseases, and its clinical manifestations include recurrent abdominal pain, bloody stools, weight loss, and systemic complications such as vomiting and malnutrition.

[0003] In recent years, the incidence of ulcerative colitis has been gradually increasing. Not only is the course of the disease long, but the mortality rate of colorectal cancer induced by it has now exceeded that of liver cancer and stomach cancer, thus attracting more and more attention. However, its pathogenesis is still unclear, but it may be closely related to genetics, environment, especially inflammation and autoimmune problems.

[0004] Most existing conventional anti-inflammatory drugs do not have good clinical efficacy for this disease. The main treatment drugs include aminosalicylic acids (such as mesalazine), glucocorticoids (dexamethasone and budesonide), immunosuppressants (such as cyclosporine), and biologics (such as adalimumab). Although they have some efficacy, they are prone to relapse, have decreased efficacy with long-term use, and are prone to serious adverse reactions. Therefore, it is of great practical significance to find a treatment for ulcerative colitis with low side effects and excellent efficacy.

[0005] Apolipoprotein J is the first secretory macromolecular chaperone discovered in the human body. It is generally believed to be closely related to a variety of physiological and pathological processes, such as inhibiting apoptosis, inactivating complement factors, lipid recycling and transport, cell membrane protection, and maintaining intercellular or cell-matrix connections. However, there are no reports on the role of apolipoprotein J in the treatment of ulcerative colitis. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides the application of recombinant apolipoprotein J in the preparation of drugs for treating ulcerative colitis. This invention has found that recombinant apolipoprotein J can alleviate the severity of colitis and has a good therapeutic effect in DSS-induced ulcerative colitis model mice.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of recombinant apolipoprotein J in the preparation of drugs for treating ulcerative colitis.

[0009] Preferably, the recombinant apolipoprotein J is a recombinant protein, comprising an amino acid primary sequence encoding recombinant apolipoprotein J, wherein the amino acid primary sequence contains two cysteine ​​residues that give the tertiary structure a pair of disulfide bonds.

[0010] Preferably, at least 95% of the primary amino acid sequence is identical to SEQ ID NO: 1. The sequence of SEQ ID NO: 1 is:

[0011] DQTVSDNELQ EMSNQGSKYV NKEIQNAVNG VKQIKTLIEK TNEERKTLLS NLEEAKKKKE 60

[0012] DALNETRESE TKLKELPGVC NETMMALWEE CKPCLKQTCM KFYARVCRSG SGLVGRQLEE 120

[0013] FLNQSSPFYF WMNGDRIDSL LENDRQQTHM LDVMQDHFSR ASSIIDELFQ DRFFTREPQD 180

[0014] TYHYLPFSLP HRRPHFFFPK SRIVRSLMPF SPYEPLNFHA MFQPFLEMIH EAQQAMDIHF 240

[0015] HSPAFQHPPT EFIREGDDDR TVCREIRHNS TGCLRMKDQC DKCREILSVD CSTNNPSQAK 300

[0016] LRRELDESLQ VAERLTRKYN ELLKSYQWKM LNTSSLLEQL NEQFNWVSRL ANLTQGEDQY 360

[0017] YLRVTTVASH TSDSDVPSGV TEVVVKLFDS DPITVTVPVE VSRKNPKFME TVAEKALQEY 420

[0018] RKKHREE 427

[0019] Preferably, the cysteine ​​is located at amino acid sites 107 and 263, or 99 and 273, or 94 and 280, or 91 and 283, or 80 and 291 of SEQ ID NO: 1. Preferably, the cysteine ​​is located at amino acid sites 107 and 263 of SEQ ID NO: 1, and amino acid sites 80, 91, 94, 99, 273, 280, 283, and 291 of SEQ ID NO: 1 are glycine or alanine.

[0020] Preferably, the recombinant apolipoprotein J is the only active ingredient in the drug for treating ulcerative colitis.

[0021] Preferably, recombinant apolipoprotein J is an intravenous preparation suitable for clinical use in the treatment of ulcerative colitis.

[0022] Preferably, the intravenous injection preparation is sodium chloride injection, glucose injection, sodium chloride-glucose injection, propylene glycol injection, or mannitol injection.

[0023] Preferably, the dosage of apolipoprotein J is 0.1-5 mg / kg / day.

[0024] This invention provides a pharmaceutical preparation for treating ulcerative colitis, wherein recombinant apolipoprotein J is contained as an active ingredient.

[0025] Preferably, the recombinant apolipoprotein J contains a pair of disulfide bonds.

[0026] Preferably, the pharmaceutical preparation is sodium chloride injection, glucose injection, sodium chloride-glucose injection, propylene glycol injection, or mannitol injection.

[0027] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to discover and confirm that recombinant apolipoprotein J can effectively alleviate DSS-induced diarrhea in mice, significantly improve the hematochezia in mice, effectively alleviate colonic shortening in mice, and effectively alleviate pathological damage to colonic tissue in mice. It has a therapeutic effect on DSS-induced ulcerative colitis in mice, providing a new application direction for recombinant ApoJ in the preparation of drugs for the treatment of ulcerative colitis. Attached Figure Description

[0028] Figure 1 This is a graph showing the colon length, weight, and colon coefficient of mice in each group in Example 1 of the present invention;

[0029] Figure 2 These are actual images of the colons of mice after dissection in each group of mice in Example 1 of this invention;

[0030] Figure 3 These are histopathological images of mice in each group after dissection in Example 1 of this invention. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The invention will be further described in detail below with reference to specific embodiments; it should be noted that these specific embodiments are illustrative rather than limiting of the invention.

[0032] Example 1: Therapeutic effect of recombinant apolipoprotein J on sodium dextran sulfate (DSS)-induced ulcerative colitis in mice.

[0033] Ulcerative colitis (UC) is a chronic inflammatory disease characterized by extensive inflammatory cell infiltration in the colonic mucosa. Patients typically present with symptoms such as abdominal pain, diarrhea, bloody and purulent stools, and tenesmus. Currently, there are various methods for preparing UC animal models, primarily including chemical induction, adoptive transfer of CD4+ T lymphocytes, gene knockout, and transgenic methods. Compared to other inducers, the colitis mouse model established by DSS induction most closely resembles human ulcerative colitis in terms of symptoms and colonic pathology, and is therefore widely used.

[0034] 1. Modeling Methods

[0035] Twenty-five female C57BL / 6J mice weighing 17-20g were randomly divided into five groups of five each: a normal control group, a model control group, a positive control group, a low-dose group, and a high-dose group. Mice in the normal control group had normal access to water, while the other mice were given free access to water containing 30g / L DSS solution. The water was changed every other day to induce a mouse model of ulcerative colitis.

[0036] 2. Test methods

[0037] Mice in the normal control group drank water normally during the experiment and were not given intravenous drug administration.

[0038] During the experiment, mice in the model control group were intravenously injected with 0.9% sodium chloride solution at a volume of 8 ml / kg, an administration rate of approximately 0.5-1 ml / min, and a frequency of once every 2 days.

[0039] Mice in the positive control group were intravenously injected with cyclosporine solution at a dose of 50 mg / kg, a concentration of 20 mg / mL, a volume of 2.5 mL / kg, and a frequency of once a day. The cyclosporine solution was prepared by adding 200 μL of cyclosporine soft capsule solution to 0.8 mL of physiological saline to prepare 1.0 mL of 20 mg / mL solution. The solution was vortexed and mixed thoroughly under light-protected conditions. After preparation, the solution was evenly dispensed into two tubes and stored at 4°C under light-protected conditions. The solution was prepared every two days.

[0040] Mice in the low-dose group were intravenously injected with recombinant apolipoprotein J solution at a dose of 0.8 mg / kg, a concentration of 0.1 mg / mL, a volume of 8 mL / kg, and a frequency of once every 2 days. Mice in the high-dose group were intravenously injected with recombinant apolipoprotein J solution at a dose of 4 mg / kg, a concentration of 0.5 mg / mL, a volume of 8 mL / kg, and a frequency of once every 2 days. The recombinant apolipoprotein J was provided by Xiamen Hongguan Biotechnology Co., Ltd., and has one disulfide bond and a concentration of 0.5 mg / mL. The corresponding amino acid sequence is as described above, that is, cysteine ​​is located at amino acid sites 107 and 263 of SEQ ID NO: 1, and glycine is located at amino acid sites 80, 91, 94, 99, 273, 280, 283, and 291.

[0041] 3. Experimental Results

[0042] 3.1 Rate of weight change

[0043] The body weight of the mice was measured and recorded every 24 hours starting from the day of disease induction (day 0), and the results are shown in Table 1.

[0044] Table 1. Changes in mouse body weight in each group during D0-D5.

[0045]

[0046] As shown in Table 1, the weight of mice in the normal control group remained stable and increased slightly; the weight of mice in the model control group decreased significantly from day 4, and decreased by about 3% after 5 days, thus verifying the effectiveness of the model; compared with the model group, the weight of mice in the positive control group, low-dose group, and high-dose group changed less on days 4-5. The weight of mice in the low-dose group was close to that of the positive control group, while the weight of mice in the high-dose group was higher than that of the positive control group on day 5, and the weight loss was less.

[0047] 3.2 DAI Score

[0048] During the experiment, the diet, stool, weight and other information were recorded daily to evaluate the disease activity index. The specific scoring criteria are shown in Table 2 and the relevant results are shown in Table 3.

[0049] Table 2. DAE Scoring Criteria

[0050]

[0051] Where DAI = (weight score + stool characteristics score + rectal bleeding score / 3), the results are shown in Table 3;

[0052] Table 3. Changes in DAI scores for each group during D0-D5.

[0053]

[0054] Table 3 shows that, compared with the normal control group, the DAI score of the model control group was significantly higher than that of the normal control group starting from D3 (P<0.05). From D4 to D5, compared with the model control group, the DAI score of the positive control group was significantly lower than that of the model control group (P<0.05). At the end of the experiment, the DAI scores of the normal control group, model control group, positive control group, low-dose group, and high-dose group were 0.00±0.00, 5.00±1.58, 3.00±0.74, 5.40±1.14, and 3.20±1.92, respectively.

[0055] 3.3 Colon coefficient

[0056] After the experiment, the dissection was performed according to SOP ANI-05-02 "Euthanasia of Laboratory Animals" and PAT-01-27 "Rodent Dissection". Before dissection, the animals were weighed, anesthetized with isoflurane, and euthanized by exsanguination through the abdominal aorta. The abdominal cavity was quickly opened, the cecum and entire colon were removed, photographs were taken, and the length of the colon and rectum was measured. The results are shown below. Figure 2 ,in Figure 2 ae represent actual colon images of the normal control group, model control group, positive control group, low-dose group, and high-dose group, respectively. After an ice bath and PBS rinsing, the colons were longitudinally dissected along the mesentery and weighed. The mice were dissected to obtain the distal end from the cecum to the rectum, and the entire length of the colon was immediately measured. The colon length and weight of each group were then calculated based on these measurements. The results are shown in [Figure number missing]. Figure 1-2 And Table 4, where “*” represents P < 0.05 compared with the model control group, “**” represents P < 0.01 compared with the model control group, and “***” represents P < 0.001 compared with the model control group.

[0057] Table 4. Colon length, weight, and colon coefficient for each group

[0058]

[0059] At the endpoint of the experiment, the colon lengths of the animals in the normal control group, model control group, positive control group, low-dose test substance group, and high-dose test substance group were 7.63±0.22 cm, 4.58±0.85 cm, 5.58±0.62 cm, 4.72±0.32 cm, and 5.47±0.25 cm, respectively; the colon weights were 230.80±40.60 mg, 231.40±44.64 mg, 206.40±18.56 mg, 214.60±18.04 mg, and 237.60±25.13 mg, respectively; and the colon densities were 30.23±5.13 mg / cm³, 50.43±1.94 mg / cm³, 37.12±2.46 mg / cm³, 45.45±2.58 mg / cm³, and 43.37±2.75 mg / cm³, respectively. mg / cm, and the gross colon scores were 0.00±0.00, 2.00±0.00, 1.60±0.55, 1.80±0.45, and 1.60±0.55, respectively.

[0060] Compared with the normal control group, the colon length of the animals in the model control group was significantly shorter than that of the normal control group (P<0.001), while the colon density and gross colon score were significantly higher than those of the normal control group (P<0.001). Compared with the model control group, the colon length of the positive control group and the high-dose test substance group was significantly greater than that of the model control group (P<0.01, P<0.05); the colon density of the positive control group, the low-dose test substance group, and the high-dose test substance group was significantly lower than that of the model control group (P<0.001, P<0.05, P<0.01).

[0061] 3.4 Histopathology

[0062] The colonic inflammation was observed under an anatomical microscope, and the gross morphological score of the colorectal mucosa was performed. The gross morphological scoring criteria and histopathological scoring criteria are shown in Tables 5-6, and the results are shown in Table 7. Figure 3 .

[0063] Table 5. Colorectal Mucosal Morphological Scoring Criteria

[0064]

[0065] Table 6 Histopathological Scoring Criteria

[0066]

[0067] Table 7 Histopathological grading and incidence in each group

[0068]

[0069] As shown in Table 7, the histopathological examination results of the colorectal tissue of 5 / 5 animals in the normal control group showed a score of 0. Figure 3-a, no inflammatory cell infiltration, intact mucosa, and normal epithelial cell morphology. Compared with the normal control group, the colorectal tissue characteristics, mucosal structure, and epithelial cell score of 5 / 5 animals in the model control group were grade 3. Figure 3 -b; Compared with the model control group, 2 / 5 animals in the positive control group had colorectal tissue characteristics, mucosal structure, and epithelial cell scores of grade 1. Figure 3 -c; 2 / 5 animals scored grade 2. Compared with the model control group, 4 / 5 animals in the low-dose group of the test substance had grade 2 scores in colorectal tissue characteristics, mucosal structure, and epithelial cells - see Figure 3 -d; 1 / 5 of the animals scored grade 3. Compared with the model control group, 2 / 5 of the animals in the high-dose group of the test substance had colorectal tissue characteristics, mucosal structure, and epithelial cell scores of grade 1, see [link to relevant documentation]. Figure 3 -e; 2 / 5 animals scored grade 2. This indicates that, under the experimental conditions, the chronic ulcerative colitis model in the C57BL / 6J mouse model control group was successfully established compared to the normal control group; compared to the model control group, the colorectal tissue characteristics, mucosal structure, and epithelial cell grading scores of the positive control group and the low and high dose groups of the test substance were all decreased.

[0070] In summary, after treatment, the high-dose group of the test substance in the model animals described in this application can improve the colon index to a certain extent. The colorectal tissue characteristics, mucosal structure and epithelial cell grading scores of the low- and high-dose groups of the test substance all decreased, suggesting that the test substance-recombinant ApoJ has a certain therapeutic effect on the DSS-induced mouse ulcerative colitis model.

[0071] Example 2: Therapeutic effect of apolipoprotein J on diglucan sulfate sodium (DSS)-induced ulcerative colitis in mice.

[0072] Modeling and animal experiments were conducted again using the same method as in Example 1, with the difference being that mice in the low-dose group were intravenously injected with recombinant apolipoprotein J solution at a dose of 4 mg / kg, a concentration of 0.4 mg / mL, an administration volume of 10 mL / kg, and a dosing frequency of once daily; mice in the high-dose group were intravenously injected with recombinant apolipoprotein J solution at a dose of 20 mg / kg, a concentration of 2 mg / mL, an administration volume of 10 mL / kg, and a dosing frequency of once daily. Apolipoprotein J was provided by Xiamen Hongguan Biotechnology Co., Ltd., at concentrations of 0.4 mg / mL and 2 mg / mL. The results are as follows:

[0073] 2.1 Rate of change in body weight

[0074] The body weight of mice was measured and recorded every 24 hours starting from the day of disease induction (day 0), and the results are shown in Table 8.

[0075] Table 8. Changes in mouse body weight in each group during D0-D5.

[0076]

[0077] As shown in Table 1, the body weight of mice in the normal control group remained relatively stable; the body weight of mice in the model control group continued to decrease, decreasing by about 5% after 5 days, thus verifying the effectiveness of the model. Compared with the model group, the body weight of mice in the positive control group, low-dose group, and high-dose group changed less on days 3-5, with the body weight of mice in the low-dose group being close to that of the positive control group.

[0078] 2.2 DAI Score

[0079] During the experiment, the diet, stool, weight and other information were recorded daily to evaluate the disease activity index. The results are shown in Table 9.

[0080] Table 9. Changes in DAI scores for each group during D0-D5.

[0081]

[0082] As shown in Table 9, compared with the normal control group, the DAI score of the model control group was significantly higher than that of the normal control group starting from D3 (P<0.05). From D4 to D5, compared with the model control group, the DAI score of the positive control group was significantly lower than that of the model control group (P<0.05). At the end of the experiment, the DAI scores of the normal control group, model control group, positive control group, low-dose group, and high-dose group were 0.60±0.55, 4.50±0.71, 2.20±0.27, 2.80±0.84, and 3.40±0.55, respectively.

[0083] 2.3 Colon coefficient

[0084] After the experiment, dissections were performed according to SOP ANI-05-02 "Euthanasia of Laboratory Animals" and PAT-01-27 "Rodent Dissection". Before dissection, the animals were weighed, anesthetized with isoflurane, and euthanized by exsanguination from the abdominal aorta. The abdominal cavity was quickly opened, and the cecum and entire colon were removed, photographed, and the length of the colon and rectum measured. The colon was rinsed thoroughly with ice-cold PBS, longitudinally dissected along the mesentery, and weighed. The mice were dissected to obtain the distal end from the cecum to the rectum, and the entire length of the colon was immediately measured for each group of mice. The colonic coefficient was calculated based on these measurements, and the results are shown in Table 10.

[0085] Table 10 Colon length, weight, and colon coefficient for each group

[0086]

[0087] At the endpoint of the experiment, the colon lengths of the animals in the normal control group, model control group, positive control group, low-dose test substance group, and high-dose test substance group were 7.29±0.25cm, 5.46±0.65cm, 5.55±0.23cm, 5.80±0.87cm, and 5.77±1.11cm, respectively, and the colon weights were 246.60±35.73mg, 284.20±26.56mg, 260.60±15.52mg, and 278.00±3mg, respectively. The values ​​were 0.07 mg and 235.60±42.35 mg, respectively. The colon density was 33.85±4.89 mg / cm, 52.30±4.86 mg / cm, 47.05±3.82 mg / cm, 48.52±5.91 mg / cm, and 41.21±5.71 mg / cm, respectively. The gross colon scores were 0.00±0.00, 2.00±0.00, 1.40±0.55, 1.40±0.55, and 1.20±0.45, respectively.

[0088] Compared with the normal control group, the colon length of the animals in the model control group was significantly shorter than that of the normal control group (P<0.05), and the colon density was significantly higher than that of the normal control group (P<0.001). Compared with the model control group, the colon density of the high-dose test substance group was significantly lower than that of the model control group (P<0.01).

[0089] 3.4 Histopathology

[0090] The condition of colonic inflammation was observed under an anatomical microscope, and the gross morphological score of the colorectal mucosa was performed. The results are shown in Table 11:

[0091] Table 11 Histopathological grading and incidence in each group

[0092]

[0093] Table 11 shows that in the normal control group, 5 out of 5 animals scored grade 0, indicating no inflammatory cell infiltration, intact mucosa, and normal epithelial cell morphology. Compared with the normal control group, in the model control group, 4 out of 5 animals scored grade 3 in colorectal tissue characteristics, mucosal structure, and epithelial cell characteristics; 1 out of 5 animals scored grade 2. Compared with the model control group, in the positive control group, 3 out of 5 animals scored grade 1 in colorectal tissue characteristics, mucosal structure, and epithelial cell characteristics; 2 out of 5 animals scored grade 2. Compared with the model control group, in the low-dose group of the test substance, 3 out of 5 animals scored grade 1 in colorectal tissue characteristics, mucosal structure, and epithelial cell characteristics; 2 out of 5 animals scored grade 2. Compared with the model control group, in the high-dose group of the test substance, 4 out of 5 animals scored grade 1 in colorectal tissue characteristics, mucosal structure, and epithelial cell characteristics; 1 out of 5 animals scored grade 2.

[0094] Therefore, it can be concluded that the ulcerative colitis model of C57BL / 6J mice was successfully established compared with the normal control group; compared with the model control group, the colorectal tissue characteristics, mucosal structure and epithelial cell grading scores of animals in the positive control group and the low and high dose groups of the test substance were all decreased.

[0095] In summary, after five consecutive days of free access to water containing 30 g / L DSS solution, and with the control group receiving normal drinking water, the model group showed weight loss, increased DAI score, shortened colon length, and increased colon density, thus establishing the model. When the test substance was administered via tail vein injection at 4 mg / kg / day and 20 mg / kg / day, the low and high doses of recombinant ApoJ improved DAI scores, colon index, colorectal tissue characteristics, mucosal structure, and epithelial cells to some extent, indicating that recombinant ApoJ has a therapeutic effect on the DSS-induced ulcerative colitis model in mice.

[0096] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. Application of recombinant apolipoprotein J in the preparation of drugs for treating ulcerative colitis.

2. The application according to claim 1, characterized in that, The recombinant apolipoprotein J is the only active ingredient in the drug for treating ulcerative colitis.

3. The application according to claim 1, characterized in that, Recombinant apolipoprotein J is an intravenous preparation suitable for clinical use in the treatment of ulcerative colitis.

4. The application according to claim 3, characterized in that, The intravenous injection preparation is sodium chloride injection, glucose injection, sodium chloride-glucose injection, propylene glycol injection, or mannitol injection.

5. The application according to claim 1, characterized in that, The dosage of apolipoprotein J is 0.1-5 mg / kg / day.

6. A pharmaceutical preparation for treating ulcerative colitis, characterized in that, It contains recombinant apolipoprotein J as an active ingredient.

7. The pharmaceutical preparation according to claim 6, characterized in that, The recombinant apolipoprotein J contains a pair of disulfide bonds.

8. The pharmaceutical preparation according to claim 6 or 7, characterized in that, The drug preparation is sodium chloride injection, glucose injection, sodium chloride-glucose injection, propylene glycol injection, or mannitol injection.

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