Application of heptamethine indole cyanine dye IR-61 in preparation of medicine for treating colitis

Abstract

The invention discloses an application of a heptamethine indole cyanine dye IR-61 in preparation of a medicine for treating colitis. The heptamethine indole cyanine dye IR-61 adopted in the invention is a small molecule substance, and has the characteristics of good targeting property, simple preparation process, low drug toxicity and the like; meanwhile, by constructing a mouse model of high-fat-induced ulcerative colitis, it is found that the IR-61 can obviously improve the survival rate of the mouse with high-fat-induced ulcerative colitis, improve local and whole body symptoms of colitis and relieve inflammatory response and intestinal injury degree, and new application of the IR-61 in the aspect of preventing and treating high-fat-related colitis is disclosed. Furthermore, the IR-61 can be used as a potential clinical treatment medicine and has a wide application prospect in the aspect of preparing the medicine for preventing and treating the colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a heptamethine indole cyanine dye IR-61 in the preparation of a drug for treating colitis. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by intestinal mucosal barrier dysfunction. UC has become a global disease with huge medical costs and is an independent risk factor for colorectal cancer. Its pathogenesis may be closely related to the increase in high-fat diet (HFD), but the pathogenesis of UC has yet to be fully elucidated. Dexran sulfate sodium salt (DSS) is a polyanion derivative. The DSS-induced colitis model is basically consistent with the symptoms, signs and pathological characteristics of human UC. The model method is simple, reproducible and maintainable, and is currently the gold standard in the field of enteritis modeling research. Studies have shown that HFD may cause intestinal mucosal barrier dysfunction and aggravate the severity of UC by causing dysbiosis of the microbiota, inducing changes in intestinal metabolites, affecting programmed cell death of intestinal epithelial cells, reducing the secretion of intestinal goblet cells and Paneth cells, destroying intercellular junctions and damaging the immune balance of the lamina propria. HFD aggravates the occurrence and development of colitis, and the pathological damage it causes is more severe and the pathogenic mechanism is more complicated. Therefore, the intervention treatment of HFD-related colitis is more difficult. At present, the only existing treatment strategies are anti-tumor necrosis factor α antibody infliximab, 5-aminosalicylic acid, corticosteroid prednisone, immunomodulators, antibiotics, probiotics and dietary nutrients, which are mainly used to suppress the patient's immune system and relieve symptoms, but have the disadvantages of poor clinical efficacy and large side effects. Therefore, it is necessary to seek new strategies for intervention in colitis, especially high-fat diet-related colitis.

[0003] Heptamethine indole cyanine dye IR-61 (hereinafter referred to as IR-61) is a new type of mitochondrial targeted near-infrared fluorescent small molecule with the functions of cell tracking, cell imaging and cell protection. IR-61 has an excitation wavelength of 782nm and an emission wavelength of 805nm. It is lipophilic and positively charged, and can automatically assemble and form spherical nanoparticles in aqueous solution. IR-61 plays an important role in improving radiation-induced lung injury, exhaustive heart injury, urination function and erectile dysfunction in diabetic rats, sepsis, etc. However, the role of this type of compound in preventing and treating colitis, especially high-fat diet-related colitis, has not been reported. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a use of a heptamethine indole cyanine dye IR-61 in the preparation of a drug for treating colitis.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] Application of heptamethine indole cyanine dye IR-61 in the preparation of drugs for treating colitis.

[0007] The structural formula of the heptamethine indole cyanine dye IR-61 is:

[0008]

[0009] Among them: C is carbon, H is hydrogen, O is oxygen, Cl is chlorine, N is nitrogen, and Br is bromine.

[0010] In certain specific embodiments, the colitis is ulcerative colitis.

[0011] Furthermore, the ulcerative colitis is colitis associated with a high-fat diet.

[0012] In certain specific embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0013] Furthermore, the pharmaceutically acceptable excipients include, but are not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants, or a mixture of several of them.

[0014] The drug can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0015] The administration route of the drug is not limited as long as it can exert the desired therapeutic or preventive effect; including but not limited to intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0016] The dosage of each injection of the drug is 1.6-2 mg / kg. Further, the dosage is the weight of the IR-61 excluding pharmaceutically acceptable excipients. The dosage of the drug varies according to age, gender, severity of the disease and personal response, route of administration, number of administrations and dosage: the administration frequency may be but is not limited to once every three days, twice a day, once a day, once a week or once a month, or the drug provided by the present invention may be administered in the form of a sustained-release preparation, in which case a less frequent administration is required; the dosage and frequency vary according to the half-life of the preparation in the user's body, and may also vary according to whether it is a preventive application or a therapeutic application.

[0017] The drug of the present invention can also be used in combination with other drugs for treating colitis. The combined use of multiple drugs can greatly increase the success rate of treatment.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] The heptamethine indole cyanine dye IR-61 used in the present invention is a small molecule substance with the characteristics of good targeting, simple preparation process, low drug toxicity, etc. At the same time, by constructing a mouse model of high-fat-induced ulcerative colitis, it was found that IR-61 can significantly improve the survival rate of mice with high-fat colitis, improve local and systemic symptoms of colitis, and reduce inflammatory response and intestinal damage, revealing a new use of IR-61 in preventing and treating high-fat-related colitis, and further indicating that IR-61 can be used as a potential clinical therapeutic drug and has broad application prospects in the preparation of drugs for preventing and treating colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0021] Figure 1 This is a result diagram showing that IR-61 improves the survival rate of HFD+DSS-induced mice in the present invention;

[0022] Figure 2 This is a result diagram showing that IR-61 reduces the degree of weight loss in mice induced by HFD+DSS in the present invention;

[0023] Figure 3 This is a result diagram showing that IR-61 in the present invention reduces the disease activity index score of HFD+DSS-induced mice;

[0024] Figure 4 This is a result diagram showing that IR-61 reduces the shortening of the colon in mice induced by HFD+DSS in the present invention;

[0025] Figure 5 This is a result diagram showing that IR-61 in the present invention alleviates the degree of colon pathological damage in mice induced by HFD+DSS;

[0026] Figure 6 This is a result diagram showing that IR-61 reduces the levels of FABP2, Lcn2, and S100A8, markers of intestinal damage in mice induced by HFD+DSS;

[0027] Figure 7 This is a graph showing the results of IR-61 reducing the levels of serum inflammatory cytokine IL-6 and chemokine KC in HFD+DSS-induced mice in the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0029] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0030] In the following examples, the experimental animals used were SPF-grade C57BL / 6J male mice, 6-8 weeks old, 18-22 grams, provided by Spav Biotechnology Co., Ltd.

[0031] The experimental reagents used were: sodium sulfate dextran (DSS) purchased from MP Biomedicals; high-fat diet purchased from Research Diets, USA; human urine and fecal occult blood test kit purchased from Nanjing Jiancheng Company; ELISA kits for FABP2, S100A8, and Lcn2 purchased from Elabscience; LEGENDplex TM MU Proinflam Chemokine Panel (13-plex) w / VbP and LEGENDplex TM MU Inflam Panel (13-plex) w / VbP kit was purchased from Biolegend USA; sterile syringes were purchased from Kindly Medical Device Co., Ltd.; surgical instruments were purchased from the Surgical Instrument Factory of Shanghai Medical Device Co., Ltd.

[0032] The experimental instruments used were: Synergy H1 microplate reader purchased from BioTek, USA, and digital pathology slide scanner purchased from Ningbo Jiangfeng Bioinformatics Technology Co., Ltd.

[0033] Example 1: IR-61 alleviates damage in HFD+DSS-induced colitis model in mice

[0034] This example provides the establishment of a high-fat diet and DSS model in mice and drug intervention, specifically:

[0035] The HFD model was established using SPF C57BL / 6J male mice fed with a high-fat diet for 6 weeks, and the colitis model was induced by free drinking water with 3% DSS solution for 1 week.

[0036] Mice were randomly divided into a high-fat diet HFD control group (HFD for short), a high-fat diet colitis HFD+DSS model group (HFD Vehicle for short), and an IR-61-treated high-fat diet colitis IR-61+HFD+DSS intervention group (HFD IR-61 for short). The HFD control group was fed a high-fat diet for 6 weeks and then drank ultrapure water for 1 week; the HFD+DSS model group was fed a high-fat diet for 6 weeks and then drank a 3% DSS solution prepared with ultrapure water for 1 week; the IR-61+HFD+DSS intervention group was fed a high-fat diet for 6 weeks, and then IR-61 was given for 2 weeks in the 4th week, and then drank a 3% DSS solution prepared with ultrapure water for 1 week in the 6th week of HFD.

[0037] The administration method of IR-61 is as follows: a certain volume of IR-61 solution prepared with PBS is intraperitoneally injected into mice at a dose of 2 mg / kg every 3 days, and the control group is intraperitoneally injected with an equal volume of PBS solution.

[0038] Example 2: Effects of IR-61 on body weight and survival rate in HFD+DSS-induced colitis model in mice

[0039] On the basis of Example 1, after DSS modeling of mice in different groups, the weight of mice was weighed and recorded every day, and the survival of mice was observed at a specific time every day until 11 days after DSS modeling, and the death of mice was plotted into a survival curve.

[0040] The results are as follows Figure 1 and Figure 2 As shown, from Figure 1 It can be seen that compared with the HFD+DSS model group, the survival curve of the IR-61+HFD+DSS intervention group shifted significantly to the right, indicating that IR-61 significantly improved the survival rate of HFD+DSS-induced mice.

[0041] from Figure 2 It can be seen that compared with the HFD+DSS model group, the slope of the body weight change curve of the IR-61+HFD+DSS intervention group was significantly reduced, suggesting that IR-61 reduced the degree of body weight loss induced by HFD+DSS in mice.

[0042] Example 3: Effect of IR-61 on the Disease Activity Index of HFD+DSS-Induced Colitis Model in Mice

[0043] On the basis of Example 1, an appropriate amount of fresh feces was placed on a clean glass slide, 2 drops of o-tolidine solution were added first, and then 2 drops of hydrogen peroxide solution were added. The time was counted and the color change of the feces was observed with the naked eye. If dark blue appeared immediately within 45 seconds, it was a strong positive (+++), if blue-green appeared immediately, it was a positive (++), if blue appeared within 30 to 60 seconds, it was a weak positive (+), and if no color appeared within 3 minutes, it was a negative (-).

[0044] At the same time, the degree of weight loss (<1%, 1%-5%, 6%-10%, 11%-20%, >20%), stool characteristics (formed stool, loose stool and not adhering to the anus, loose stool and adhering to the anus) and stool bleeding (occult blood negative, occult blood positive, macroscopic bloody stool) of each group of mice were evaluated, and each group of mice was evaluated and recorded as 0, 1, 2, 3, 4 points (weight), 0, 2, 4 points (stool characteristics), 0, 2, 4 points (stool blood). The sum of the scores of the three indicators was calculated, DAI = the total score of weight loss, stool characteristics, and stool blood.

[0045] The results are as follows Figure 3 As shown in the figure, it can be seen that at different times after DSS modeling, the disease activity index score of the IR-61+HFD+DSS intervention group was significantly lower than that of the HFD+DSS model group, suggesting that IR-61 reduces the disease activity index score of HFD+DSS-induced mice.

[0046] Example 4: Effect of IR-61 on colonic histopathological damage in HFD+DSS-induced colitis model in mice

[0047] On the basis of Example 1, 0.5 cm circular tissue near the anus was cut along the short axis of the colon, immersed in 4% paraformaldehyde solution for fixation, and then embedded, sliced, and HE stained. The stained colon tissue sections were observed under a light microscope.

[0048] The results are as follows Figure 4 and Figure 5 As shown, from Figure 4 It can be seen that compared with the HFD control group, the colon length of the mice in the HFD+DSS model group was significantly shortened, and the IR-61+HFD+DSS intervention group reversed the degree of colon shortening, indicating that IR-61 can alleviate the shortening of the colon length induced by HFD+DSS in mice;

[0049] Among them from Figure 5It can be seen that compared with the HFD control group, the colon epithelial crypt structure of mice in the HFD+DSS model group was extensively destroyed, goblet cells and epithelial cells were lost, a large number of inflammatory cells infiltrated, and the muscle layer was thickened after DSS modeling. The above pathological damage in the IR-61+HFD+DSS intervention group was significantly improved, indicating that IR-61 alleviates the degree of colon pathological damage induced by HFD+DSS in mice.

[0050] Example 5: Effect of IR-61 on intestinal damage markers in HFD+DSS-induced colitis model in mice

[0051] On the basis of Example 1, mouse serum was collected and tested by enzyme-linked immunosorbent assay. Standard working solution or sample was added to the corresponding plate wells, and incubated at 37°C for 90 minutes; after discarding the liquid in the plate, 100 μl of biotinylated antibody working solution was immediately added, and incubated at 37°C for 60 minutes; the liquid in the plate was discarded, and the plate was washed 3 times; 100 μl of HRP enzyme conjugate working solution was added to each well, incubated at 37°C for 30 minutes, the liquid in the plate was discarded, and the plate was washed 5 times; 90 μl of substrate solution was added to each well, and incubated at 37°C for about 15 minutes; 50 μl of stop solution was added to each well; reading was immediately performed at a wavelength of 450 nm, and the data was processed.

[0052] The results are as follows Figure 6 As shown in the figure, compared with the HFD+DSS model group, the elevated levels of intestinal injury markers FABP2, Lcn2, and S100A8 in the IR-61+HFD+DSS intervention group were significantly reduced, indicating that IR-61 can reduce the elevation of intestinal injury markers induced by HFD+DSS in mice.

[0053] Example 6: Effects of IR-61 on serum inflammatory and chemokines in HFD+DSS-induced colitis model in mice

[0054] Based on Example 1, mouse serum was collected and tested using a multifactor kit. The components in the kit were restored to room temperature, 25 μl of Assay Buffer was added to each sample tube, 25 μl of Mitrix B / C was added to each standard tube, 25 μl of each standard was added to the corresponding standard tube, 25 μl of each sample was added to the corresponding sample tube, and 25 μl of mixed beads was added to each tube, and the final volume of each tube was 75 μl. Protect from light, shake the EP tube at 800rpm, incubate at room temperature for 2h; centrifuge at 1000g for 5min, discard the supernatant; add 200μl 1×wash buffer, shake and wash, centrifuge at 1000g for 5min, discard the supernatant; add 25μl detection antibody to each tube; protect from light, shake the EP tube at 1000rpm, incubate at room temperature for 1h; do not wash, directly add 25μl SA-PE to each tube; protect from light, shake the EP tube at 1000rpm, incubate at room temperature for 30min; centrifuge at 1000g for 5min, carefully aspirate and discard 50μl supernatant with a pipette tip; add 200μl 1×wash buffer to each tube, vortex beads, centrifuge at 1000g for 5min, discard the supernatant; add 200μl 1×wash buffer to each tube. Resuspend beads; detect on the machine.

[0055] The results are as follows Figure 7 As shown in the figure, compared with the HFD+DSS model group, the elevated levels of serum inflammatory cytokine IL-6 and chemokine KC in the IR-61+HFD+DSS intervention group were significantly reduced, indicating that IR-61 can reduce the increase of serum inflammatory cytokines and chemokines in mice induced by HFD+DSS.

[0056] The present invention constructs a mouse model of ulcerative colitis induced by high fat, and finds that after intervention with IR-61, the clinical symptom scores and pathological damage degree of colitis in mice induced by high fat diet and DSS can be alleviated, the survival rate can be improved, the decrease in colon length and body weight can be reduced, and the increase in intestinal damage markers and inflammatory cytokines can be reduced. That is, the present invention discovers and verifies the new use of IR-61 as a new drug for treating colitis, especially high fat diet-related colitis.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Application of heptamethine indole cyanine dye IR-61 in the preparation of drugs for the treatment of colitis.

2. The use according to claim 1, characterized in that: The colitis is ulcerative colitis.

3. The use according to claim 2, characterized in that: The ulcerative colitis is a high-fat diet-related colitis.

4. The use according to claim 3, characterized in that: The structural formula of the heptamethine indole cyanine dye IR-61 is:

5. The use according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients include, but are not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants, or a mixture of several of them.

7. The use according to claim 3, characterized in that: The administration route of the drug includes, but is not limited to, intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

8. The use according to claim 3, characterized in that: The drug is injected in a dosage of 1.6-2 mg / kg per time.

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