Application of 7-ketodeoxycholic acid in treatment of intestinal mucosa defect related diseases

Abstract

The invention discloses application of 7-ketodeoxycholic acid in treating intestinal mucosa defect related diseases, and belongs to the field of intestinal mucosa repair. The 7-ketodeoxycholic acid can be used for preparing medicines for treating intestinal mucosal lesion related diseases, wherein the intestinal mucosal lesion related diseases comprise ulcerative colitis, Crohn's disease, irritable bowel syndrome, infectious enteritis, intestinal radiation injury and intestinal mucosal lesion caused by medicines; wherein the 7-ketodeoxycholic acid directly promotes intestinal mucosa healing by inducing endoplasmic reticulum calcium release, and improves colitis diseases and mucosa defects of a far-end colonic mucosa trauma model mouse caused by DSS and biopsy forceps.

Description

Technical Field

[0001] The present invention belongs to the field of intestinal mucosal repair, and specifically relates to the application of 7-ketodeoxycholic acid in treating diseases related to intestinal mucosal defects. Background Art

[0002] The intestinal mucosa is an important barrier in the innermost layer of the intestine, and plays a key role in maintaining the stability of the intestinal environment and immune function. Intestinal mucosal damage is an important pathological basis for a variety of intestinal diseases such as ulcerative colitis, Crohn's disease, and irritable bowel syndrome.

[0003] Although commonly used drugs in clinical practice can relieve the symptoms of patients with the above-mentioned intestinal mucosal damage-related diseases, they cannot completely repair the damaged intestinal mucosa. Incomplete mucosal repair causes the patient's intestinal mucosa to remain in a state of damage, and the disease recurs or even worsens. Studies have shown that promoting intestinal mucosal healing can help reduce the surgical rate, recurrence rate, hospitalization rate and colorectal cancer risk of patients with inflammatory bowel disease (including ulcerative colitis and Crohn's disease). Mucosal healing has become the ultimate goal of treating intestinal mucosal damage-related diseases such as ulcerative colitis. Accelerating mucosal healing helps to reestablish intestinal epithelial homeostasis and prevent disease progression and recurrence. Mucosal healing promoters have important therapeutic value.

[0004] At present, the clinical treatment of inflammatory bowel disease mainly relies on biological agents such as 5-aminosalicylic acid, corticosteroids and TNF-α monoclonal antibodies. These drugs relieve inflammation, inhibit immune response, improve disease symptoms, and then reduce mucosal damage, but they themselves have no direct effect on promoting intestinal mucosal healing.

[0005] So far, no drug that directly promotes intestinal mucosal healing has been approved for marketing, and some candidate drugs have shown potential in the preclinical and clinical trial stages. A number of randomized clinical trials have shown that recombinant growth factors have a positive effect on mucosal healing. For example, recombinant human growth hormone can promote mucosal healing in patients with Crohn's disease, and recombinant human epidermal growth factor and recombinant human keratinocyte growth factor 2 can promote mucosal healing in patients with ulcerative colitis. However, these growth factors are prone to cause side effects such as edema and headache. In addition, in the context of chronic inflammation, they may induce abnormal proliferation of epithelial cells and increase the risk of cancer in patients, thus limiting their clinical application.

[0006] In addition, ATH-063 is a small molecule inhibitor targeting histone methyltransferase EHMT2 developed by Athos, Australia, and is still in Phase I clinical development. It is reported that the drug can accelerate colon mucosal healing through anti-inflammatory effects and upregulation of tight junction protein expression. MT-5745 is a double-stranded RNA targeting the carbohydrate sulfotransferase 15 gene developed by Mitsubishi Pharma of Japan, designed to treat mucosal lesions in patients with refractory ulcerative colitis and Crohn's disease. However, the clinical trials of the drug have stalled because its efficacy was not significantly different from that of placebo.

[0007] In summary, although recombinant growth factor drugs have shown certain efficacy in promoting mucosal healing, their side effects and potential carcinogenic risks limit further development. Other candidate drugs have either stopped clinical trials due to insufficient efficacy or are still in the early stages of research and development. Therefore, finding new targets and drugs to effectively promote mucosal healing is a problem that needs to be solved urgently. Summary of the invention

[0008] In view of the deficiencies of the prior art, the purpose of the present invention is to provide the use of 7-ketodeoxycholic acid in the treatment of intestinal mucosal defect-related diseases, thereby solving the problems in the prior art.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] Application of 7-ketodeoxycholic acid in the preparation of medicines for treating diseases related to intestinal mucosal defects.

[0011] Furthermore, the diseases related to intestinal mucosal defects include: ulcerative colitis, Crohn's disease, irritable bowel syndrome, infectious enteritis, intestinal radiation damage and intestinal mucosal damage caused by drugs.

[0012] Furthermore, the dosage forms of the drug include tablets, pills, capsules, ointments, powders, solutions, granules, suspensions, injections, sustained-release preparations and controlled-release preparations.

[0013] Furthermore, the effective dose of 7-ketodeoxycholic acid is 25-100 mg / kg.

[0014] Furthermore, the drug also includes excipients, which include: one or more of pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, wetting agents, binders, disintegrants, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents and diluents.

[0015] A medicine for treating diseases related to intestinal mucosal defects, comprising a sole active ingredient: 7-ketodeoxycholic acid.

[0016] Beneficial effects of the present invention:

[0017] 1. The present invention applies 7-ketodeoxycholic acid to drugs for treating diseases related to intestinal mucosal damage, opens up new uses for 7-ketodeoxycholic acid, and provides a new option for developing drugs that promote mucosal healing.

[0018] 2. 7-Ketodeoxycholic acid can promote colon mucosal healing by inducing endoplasmic reticulum calcium release in colon epithelial cells, improve mucosal defects and colonic inflammation in mice with distal colon mucosal trauma caused by DSS and biopsy forceps, and has good application prospects for the development of therapeutic drugs for diseases related to intestinal mucosal injury.

[0019] 3. 7-Ketodeoxycholic acid is a secondary bile acid and an endogenous metabolite in the body with high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a quantitative analysis chart of serum 7-KDCA in the control group and patients with ulcerative colitis;

[0022] Figure 2 This is the quantitative analysis of 7-KDCA in serum and colon tissue of the control group and colitis model mice;

[0023] Figure 3 This is a graph showing the effect of 7-KDCA on the survival rate, disease activity index, and colon length of colitis model mice;

[0024] Figure 4 This is a graph showing the effect of 7-KDCA on colonoscopic scores, histological damage, and intestinal permeability in colitis model mice;

[0025] Figure 5 This is a graph showing the effect of 7-KDCA on wound healing in mice with biopsy-induced intestinal mucosal injury model;

[0026] Figure 6 This is a graph showing the effect of 7-KDCA on calcium levels in intestinal epithelial cells;

[0027] Figure 7 It is the structural formula of 7-KDCA. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] 7-Ketodeoxycholic acid (7-KDCA) structure Figure 7 As shown, the use of 7-KDCA is described below from the following examples.

[0030] Example 1

[0031] This example reflects that the serum level of 7-ketodeoxycholic acid (7-KDCA) in patients with ulcerative colitis decreases;

[0032] A total of 8 ulcerative colitis patients and 8 healthy subjects were recruited from the gastroenterology clinic of a traditional Chinese medicine hospital. Fasting blood was collected from all subjects. After coagulation, the blood was centrifuged at 3500rpm for 5 minutes to obtain serum. 100μL of serum was taken, 500μL of pre-cooled methanol and 10μL of 200ng / mL isotope internal standard were added, vortexed for 60s, and allowed to stand at -20℃ for 1h. Centrifuged (12000rpm, 4℃) for 20min, the supernatant was taken, and mass spectrometry quantitative analysis was performed. The experimental results are shown in Figure 1 , serum 7-KDCA levels in patients with ulcerative colitis were reduced by 61.2% compared with healthy subjects.

[0033] The experimental results showed that the serum 7-KDCA levels in patients with ulcerative colitis were significantly lower than those in healthy subjects.

[0034] Example 2

[0035] This example reflects that the 7-KDCA levels in serum and colon tissue of colitis model mice decreased;

[0036] Mice were randomly divided into a normal group and a DSS group according to body weight. The normal group had free drinking water every day, while the mice in the DSS group were given 3.5% DSS solution for 5 consecutive days, followed by double distilled water for 4 days. The day of modeling was recorded as day 1. Mice were dissected during the induction period (day 3), acute period (day 6), and recovery period (day 9), and samples were taken to detect relevant indicators. 25±0.1 mg of terminal colon tissue was placed in a 2mL EP tube, 1000μL of methanol-acetonitrile-water (2:2:1) was added, and an appropriate amount of magnetic beads was added. The tissue was homogenized by a tissue grinder (frequency 60Hz, running time 60s, interval 10s, 5 cycles), ultrasonic extraction for 30min, and centrifugation (12000rpm, 4℃) for 10min. The supernatant was dried at 37℃ using a vacuum concentrator dryer. 50μL of serum was taken, 200μL of pre-cooled methanol was added, vortexed for 60s, and allowed to stand at -20℃ for 1h. Centrifuge (12000rpm, 4℃) for 20min, take the supernatant and freeze-dry. The dried sample was re-dissolved with 200μL of 50% methanol (containing the internal standard 2μg / mL 2-chloro-L-phenylalanine), centrifuged (12000rpm, 4℃) for 10min, and 100μL of the supernatant was taken for LC-MS analysis. The experimental results are shown in Figure 2 In (a) and (b), (a) shows the changes in the concentration of 7-KDCA in the serum of colitis model mice during the induction period (day 3), acute period (day 6), and recovery period (day 9); (b) shows the changes in the concentration of 7-KDCA in the colon tissue of colitis model mice during the induction period (day 3), acute period (day 6), and recovery period (day 9). Based on the above results, it can be seen that the 7-KDCA level began to gradually decrease as early as the third day after DSS administration, reaching the lowest point on the sixth day; after removing DSS, it returned to a near normal level.

[0037] The experimental results showed that compared with the control group, the 7-KDCA levels in the serum and colon tissue of colitis model mice were significantly reduced.

[0038] Example 3

[0039] This example reflects the improvement effect of 7-KDCA on DSS-induced colitis in mice

[0040] Mice were randomly divided into a normal group, a DSS group, and a 7-KDCA group (50 mg / kg) according to their body weight. Mice in the DSS group and the drug-treated group were given 3.5% DSS solution for 5 consecutive days, and then switched to double distilled water for drinking for 5 days. The day of modeling was recorded as day 1. The drug-treated group began to be gavaged for 5 consecutive days starting on the 6th day. The mice were killed on the 10th day, dissected, and the colon was removed, its natural length was measured, and photographed for record. The experimental results are shown in Figure 3In (a) to (d), (a) shows the survival rate of mice in each experimental group, (b) shows the disease activity index score of mice in each experimental group, (c) shows the colon length of mice in each experimental group, and (d) shows the colon length measurement results of mice in each experimental group. From the above results, it can be seen that 7-KDCA significantly improves the survival rate of colitis mice, reduces the disease activity index, and inhibits the shortening of colon length.

[0041] Experimental results showed that 7-KDCA could improve DSS-induced colitis in mice.

[0042] Example 4

[0043] This example reflects the promoting effect of 7-KDCA on the healing of colon mucosa in mice with DSS-induced colitis;

[0044] Mice were randomly divided into a normal group, a DSS group, and a 7-KDCA group (50 mg / kg) according to their body weight. Mice in the DSS group and the drug-treated group were given 3.5% DSS solution for 5 consecutive days, followed by double distilled water for drinking for 5 days. The day of modeling was recorded as day 1. The drug-treated group was given oral administration for 5 consecutive days starting on the 6th day, and endoscopic monitoring was performed on the 10th day. Endoscopic scoring was performed based on perianal lesions, intestinal wall transparency, intestinal bleeding, focal lesions (such as erosion, ulcers), intestinal stenosis, masses, tears / perforations, and death during endoscopic examination. The mice were then killed, dissected, and the colon was removed. By rolling up the entire colon like a "Swiss roll", paraffin-embedded sections (6 μm thick) were prepared for H&E staining to quantify colon mucosal damage. The percentage of injury / ulceration was calculated as the ratio of the length of the injury / ulcer area (≥50% crypt loss) to the length of the entire colon. In addition, mice were fasted for 6 h and given FITC (fluorescein isothiocyanate)-dextran solution (440 mg / kg) by gavage. Mice were killed 4 h later and serum was collected. Serum FITC-dextran was detected on a spectrophotometer at 485 / 530 nm, and a standard curve was obtained by serially diluting FITC-dextran in PBS. The experimental results are shown in Figure 4 (a) to (d), where (a) shows the colonoscopic photos of mice in each experimental group on the 6th and 10th days, respectively, (b) shows the colonoscopic scoring results of mice in each experimental group, (c) shows the percentage of colon injury length of mice in each experimental group, and (d) shows the serum FITC concentration. FITC is fluorescein isothiocyanate, which is commonly used to evaluate intestinal permeability. The results showed that 7-KDCA significantly reduced the endoscopic scores of colitis mice, alleviated colon mucosal ulcer damage, and reduced intestinal permeability.

[0045] The experimental results showed that 7-KDCA promoted the healing of colon mucosa in mice with DSS-induced colitis.

[0046] Example 5

[0047] This example reflects the promoting effect of 7-KDCA on the healing of intestinal mucosa in mice with biopsy-induced colon mucosal injury;

[0048] Biopsy forceps were used to induce colon mucosal injury in mice. The size of mucosal wounds was quantified 24 hours after injury. 7-KDCA (50 mg / kg) was administered orally to mice, and the wound healing rate was detected 72 hours after injury. Figure 5 In (a) and (b), (a) shows wound images of mice in the control group and 7-KDCA-treated group at 24h and 72h after biopsy-induced colon mucosal injury, and (b) shows quantitative data of the image acquisition of (a). The data show that 7-KDCA significantly accelerates colon mucosal wound healing 72h (or day 3) after biopsy (control group: 27.85±6.63%; 7-KDCA group: 58.28±11.31%).

[0049] The experimental results showed that 7-KDCA significantly promoted the healing of intestinal mucosa in mice with biopsy-induced colon mucosal injury.

[0050] Example 6

[0051] This example reflects the effect of 7-KDCA on increasing the intracellular calcium level of intestinal epithelial cells

[0052] NCM-460 cells (1×10 5 Cells / mL) were inoculated in 48-well cell culture plates, and incubated with or without 7-KDCA (dissolved in 1% serum medium). Then 1 μM Fluo 3AM (dissolved in PBS) was added to the cells and incubated for 30 min. After washing with PBS, Hoechst (dissolved in PBS) was added and incubated at 37°C for 20 min. After washing with PBS 3 times, the cells were photographed and recorded, and the fluorescence was analyzed and recorded using Image J. The experimental results are shown in Figure 6 , 7-KDCA increased the intracellular calcium level of NCM-460 in a concentration-dependent manner.

[0053] The experimental results showed that 7-KDCA increased the intracellular calcium level of intestinal epithelial cells.

[0054] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

Application of 1.7-ketodeoxycholic acid in the preparation of drugs for treating diseases related to intestinal mucosal defects.

2. The use according to claim 1, characterized in that: The diseases related to intestinal mucosal defects include: ulcerative colitis, Crohn's disease, irritable bowel syndrome, infectious enteritis, intestinal radiation damage and intestinal mucosal damage caused by drugs.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include tablets, pills, capsules, ointments, powders, solutions, granules, suspensions, injections, sustained-release preparations and controlled-release preparations.

4. The use according to claim 1, characterized in that: The effective dosage of 7-ketodeoxycholic acid is 25-100 mg / kg.

5. The use according to claim 1, characterized in that: The drug also includes excipients, which include: one or more of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, wetting agents, binders, disintegrants, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents and diluents.

6. A drug for treating diseases related to intestinal mucosal defects, characterized in that: The drug comprises a sole active ingredient: 7-ketodeoxycholic acid.

7. A drug for treating diseases related to intestinal mucosal defects according to claim 6, characterized in that: The dosage forms of the drug include tablets, pills, capsules, ointments, powders, solutions, granules, suspensions, injections, sustained-release preparations and controlled-release preparations.

8. The drug for treating diseases related to intestinal mucosal defects according to claim 6, characterized in that: The effective dosage of 7-ketodeoxycholic acid is 25-100 mg / kg.

9. The drug for treating diseases related to intestinal mucosal defects according to claim 6, characterized in that: The drug also includes excipients, which include: one or more of pharmaceutically acceptable solvents, solubilizers, co-solvents, emulsifiers, wetting agents, binders, disintegrants, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, inclusion agents and diluents.

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