Preparation for treating colon inflammation and preparation method thereof

Abstract

The present application provides a preparation that can be used for colitis, comprising arctiin; chitosan; and arctiin and chitosan connected via a diselenide bond. A method for preparing the aforementioned preparation is also provided. Compared to other existing technologies with complex preparation processes and inconvenient administration methods, the preparation method of the present application is simple, has a stable yield, and is convenient and quick to synthesize in only two steps. The prepared hydrogel also uses a safe and non-toxic natural non-toxic cross-linking agent as a cross-linking agent, has good efficacy, can be administered orally, and has better patient compliance than rectal administration. It has significant advantages and extremely high development potential for clinical development and use.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a preparation for treating colon inflammation and a preparation method thereof. Background Art

[0002] Ulcerative colitis, an inflammatory bowel disease (IBD), is a chronic, nonspecific inflammatory disease of the colon and rectum with an unclear etiology. Lesions in ulcerative colitis are limited to the large intestinal mucosa and submucosa, primarily located in the sigmoid colon and rectum, but can also extend to the descending colon or even the entire colon. Ulcerative colitis has a long course and often recurs, with its incidence increasing annually both domestically and internationally. Multiple factors, including genetic susceptibility, epithelial barrier defects, immune dysregulation, and environmental factors, all play a significant role in the pathogenesis of ulcerative colitis.

[0003] Currently, the most effective oral medication, 5-aminosalicylic acid enteric-coated tablets, uses enteric-coated capsules for intestinal drug release. These capsules utilize the properties of enteric-coated capsules to achieve targeted drug release within the intestine. However, these capsules target the entire intestine and are unable to precisely deliver the drug to affected colon ulcers. Other routes of administration, such as rectal administration, can precisely target the colon, but they pose challenges such as poor compliance and inconvenience. Summary of the Invention

[0004] The technical problems to be solved by this application are:

[0005] Existing technical methods for treating ulcerative colitis have problems with either inaccurate drug release or poor compliance.

[0006] Technical means to solve the problem:

[0007] Arctigenin (ATG), a lignan compound and the main active ingredient in the traditional Chinese medicine (Burdock), possesses antiviral and anti-inflammatory properties. ATG is known to inhibit the expression of several genes involved in inflammation in the colon, including TNF-α, IL-6, macrophage inflammatory protein, monocyte chemoattractant protein, immunoglobulin adhesion molecule, and vascular adhesion molecule. Therefore, ATG has positive clinical implications for the treatment of ulcerative colitis.

[0008] In order to achieve targeted delivery of ATG to the affected area during the treatment of ulcerative colitis, so that the ATG drug can exert a specific effect on the affected area of ​​colon ulcers and can be continuously released at the affected area to achieve a sustained-release effect, the present application provides a method for targeted delivery of ATG. That is, since the occurrence of colon inflammation is closely related to the overexpression of reactive oxygen species in the colon tissue microenvironment, the present application provides a preparation that uses a diselenide bond to carry ATG. The preparation has ROS responsiveness. In the colon tissue microenvironment with high reactive oxygen species, the diselenide bond of the preparation will break, thereby releasing ATG, which can accurately produce an effect at the colon ulcer.

[0009] Specifically, this application addresses the aforementioned issues:

[0010] Provided is a preparation useful for colon inflammation, comprising:

[0011] Arctigenin;

[0012] Chitosan;

[0013] The arctigenin is connected to chitosan via a diselenide bond.

[0014] Optionally, the substitution ratio of arctigenin to chitosan in the preparation is (0.25-4):1 or other reasonable substitution ratios.

[0015] Optionally, a cross-linking agent is further included, and the cross-linking agent is a natural non-toxic cross-linking agent.

[0016] Optionally, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 5-10:0.1.

[0017] Optionally, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 5-10:0.1.

[0018] Optionally, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 5-10:0.1.

[0019] In addition, a preparation for treating colon inflammation is provided, which is characterized by comprising:

[0020] Arctigenin;

[0021] Chitosan;

[0022] cross-linking agent;

[0023] The arctigenin and chitosan are connected via a diselenide bond; and the cross-linking agent is a natural non-toxic cross-linking agent.

[0024] Optionally, the dissolution rate of the preparation is 25-35%.

[0025] Optionally, the degree of swelling of the preparation is not less than 3000%.

[0026] In addition, a method for preparing a preparation that can be used for colon inflammation is also provided, which comprises:

[0027] S1, mixing appropriate amounts of selenomalonic acid and 1-hydroxybenzotriazole and dissolving the mixture in a mixed solution containing dichloromethane and DMSO;

[0028] S2, adding an appropriate amount of arctigenin to the solution obtained in S1 and reacting for (12-48) hours to obtain a reaction solution;

[0029] S3, rotary evaporating the reaction solution obtained in S2, adding DMSO to dissolve it, and then sequentially adding appropriate amounts of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), ice-bathing for (1-5) hours, and stirring for activation for (24-72) hours to prepare an active ester;

[0030] S4, mixing the active ester obtained in S2 with an appropriate amount of chitosan, adding DMSO to dissolve the mixture, and reacting for 24 to 48 hours to obtain the preparation.

[0031] According to the above technical solution, it can be seen that this application has the following advantages:

[0032] Compared with other existing technologies with complex preparation processes and inconvenient administration methods, the preparation method of the present application is simple, with stable yields and only two steps of synthesis, which is convenient and fast. The prepared hydrogel also uses a safe and non-toxic natural non-toxic cross-linking agent as a cross-linking agent, has good efficacy, can be administered orally, and has better patient compliance than rectal administration. It has significant advantages and extremely high development potential for clinical development and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] This application also provides drawings related to the technical solutions provided by this application to illustrate the technical solutions of this application. The purpose of the drawings and descriptions is only to more clearly describe this application and should not be regarded as limiting the scope of protection claimed in this application.

[0034] Figure 1 The gel state of genipin and chitosan at different concentration ratios;

[0035] Figure 2 The dissolution curves of hydrogels with different substitution ratios of ATG and chitosan;

[0036] Figure 3 The swelling curves of hydrogels with different substitution ratios of ATG and chitosan;

[0037] Figure 4 The rheological property curve of the preparation of the present application is shown;

[0038] Figure 5 The rheological property curve of the gel including only chitosan as a control group is shown;

[0039] Figure 6 and Figure 7 The three-dimensional network structure of the preparation of the present application under a SEM scanning electron microscope is shown;

[0040] Figure 8 The in vitro release dissolution curve of the preparation of the present application;

[0041] Figure 9 Shown are the effects of the formulations of the present application on cellular ROS levels in Caco-2 cells;

[0042] Figure 10 The DAI scores of ulcerative colitis mice modeled with DSS within seven days of administration are shown;

[0043] Figure 11 The figure shows the comparison results of colon length of each group of DSS-induced ulcerative colitis mice;

[0044] Figure 12 The comparative results of colon tissue staining of DSS-induced ulcerative colitis mice are shown;

[0045] Figure 13 The figure shows the expression level of occludin-1 protein in each group of DSS-induced ulcerative colitis mice. DETAILED DESCRIPTION

[0046] The following is a detailed description of one (or more) implementation methods of the present application, the purpose of which is to clearly and completely illustrate the essence of the technical solution provided by the present application.

[0047] Preparation method of the preparation of the present application

[0048] Weigh selenomalonic acid and 1-hydroxybenzotriazole (HOBt) into a round-bottom flask, add them to a mixed solution of dichloromethane (DCM) and DMSO, and wait until they are completely dissolved. Then weigh an appropriate amount of arctigenin (ATG) and dissolve it in DCM. Slowly add it dropwise to the round-bottom flask using a separatory funnel. The reaction molar ratio of the three is 1:1~2:1~2. React for (24~48) hours to obtain a reaction solution.

[0049] The reaction solution was rotary evaporated, weighed, and redissolved in 1-20 ml of DMSO. HOBt and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were then added sequentially at a molar ratio of 1:1-2:1-2. The mixture was stirred in an ice bath and activated for 1-5 h to prepare an active ester. Chitosan was added to 1-5 ml of the active ester at different ATG:CS substitution ratios (1:1, 1:2, 1:5, 1:10, and 1:20) and allowed to react for 24-48 h.

[0050] This application

[0051] In one aspect, the present application provides a preparation that can be used for colon inflammation, comprising:

[0052] Arctigenin;

[0053] Chitosan;

[0054] The arctigenin is connected to chitosan via a diselenide bond.

[0055] In some embodiments, the substitution ratio of arctigenin to chitosan in the preparation is (0.25-4):1 or other reasonable substitution ratios.

[0056] In some embodiments, a cross-linking agent is further included, and the cross-linking agent is a natural non-toxic cross-linking agent.

[0057] In some embodiments, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 6-9:0.1.

[0058] In some embodiments, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 7-8:0.1.

[0059] In some embodiments, the concentration ratio of the sodium alginate, genipin or other natural non-toxic cross-linking agent to the chitosan is 7.5:0.1.

[0060] On the other hand, the present application also provides a preparation that can be used for colon inflammation, comprising:

[0061] Arctigenin;

[0062] Chitosan;

[0063] cross-linking agent;

[0064] The arctigenin is connected to chitosan via a diselenide bond; and the cross-linking agent is sodium alginate, genipin or other natural non-toxic cross-linking agents.

[0065] In some embodiments, the formulation has a dissolution rate of 25-35%.

[0066] In some embodiments, the degree of swelling of the formulation is no less than 3000%.

[0067] On the other hand, the present application also provides a method for preparing a preparation that can be used for colon inflammation, comprising:

[0068] S1, mixing appropriate amounts of selenomalonic acid and 1-hydroxybenzotriazole and dissolving the mixture in a mixed solution containing dichloromethane and DMSO;

[0069] S2, adding an appropriate amount of arctigenin to the solution obtained in S1 and reacting for (24-48) hours to obtain a reaction solution;

[0070] S3, rotary evaporating the reaction solution obtained in S2, adding DMSO to dissolve it, and then sequentially adding appropriate amounts of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), ice-bathing for (1-5) hours, and stirring for activation for (24-72) hours to prepare an active ester;

[0071] S4, mixing the active ester obtained in S2 with an appropriate amount of chitosan, adding DMSO to dissolve the mixture, and reacting for 24 to 48 hours to obtain the preparation.

[0072] Experimental part

[0073] 1. NMR confirmation and degree of substitution determination

[0074] Proton NMR spectroscopy was used to confirm the synthesis of the formulation described herein and to determine its degree of substitution. Integration of the NMR spectra of ATG-Se-Se-CS with varying degrees of substitution revealed a 4.18% content when the ratio of ATG:CS = 2:1 was used.

[0075] 2. Investigation of hydrogel formation

[0076] In order to better prepare suitable hydrogels, we conducted experiments on different concentrations of genipin and chitosan to screen out the best hydrogel formation conditions. At 50℃, we adjusted the concentrations of genipin and chitosan and observed the gel state. The results are as follows: Figure 1 shown.

[0077] From left to right, the concentrations of CS and JNP are:

[0078] No. 1: CS 5mg / ml, JNP 0.1mg / ml

[0079] No. 2: CS 7.5mg / ml, JNP 0.1mg / ml

[0080] No. 3: CS 10mg / ml, JNP 0.1mg / ml

[0081] No. 4: CS 5mg / ml, JNP 0.2mg / ml

[0082] No. 5: CS 7.5mg / ml, JNP 0.2mg / ml

[0083] No. 6: CS 10mg / ml, JNP 0.2mg / ml

[0084] In order to maintain a certain gel viscosity and a certain fluidity at the same time, it can be seen that No. ② is the best.

[0085] 3. Drug solubility prescription screening

[0086] The formulations described herein, each with a different substitution ratio of ATG to chitosan, were quantitatively added to a centrifuge tube containing 40 mg of ATG. 40 ml of PBS buffer containing 4% SDS was then added as the dissolution medium. UV absorbance was measured at 1, 2, 3, and 4 hours. The concentration was calculated using the standard curve y = 0.4472x - 0.0226, and the dissolution rate was then calculated. The results are shown in Figure 1. Figure 2 shown.

[0087] according to Figure 2 It can be seen that when ATG:CS=2:1, the release effect of the preparation described in the present application is better.

[0088] 4. Drug Hydrogel Swelling Assay

[0089] The swelling degree of the preparations described in this application with different substitution ratios of ATG and chitosan was measured. The higher the swelling degree, the better the water retention of the hydrogel. Figure 3 shown.

[0090] according to Figure 3 It can be seen that when ATG:CS=2:1, the swelling degree of the preparation described in the present application is the highest.

[0091] 5. Investigation of rheological properties of drug hydrogels

[0092] The formulation described in this application (i.e., ATG-Se-Se-CS-Gel) and the gel comprising only chitosan (i.e., CS-Gel) were subjected to rheometer measurements, and the results were as follows: Figure 4 and Figure 5 shown.

[0093] according to Figure 4 and Figure 5 The results show that the modulus crossover point of the gel containing only chitosan (CS-Gel) is greater than that of the preparation described in the present application (ATG-Se-Se-CS-Gel). Therefore, it can be seen that the viscosity of the preparation described in the present application is better and it is more conducive to adhesion to the colon.

[0094] 6. Characterization of Hydrogels

[0095] The three-dimensional structure of the preparation of the present application was observed by SEM scanning electron microscopy. Figure 6 and Figure 7 As shown. Figure 6 and Figure 7 It can be seen that the preparation of the present application presents a three-dimensional network space structure, proving that the hydrogel has been formed and has a good morphology and a certain swelling space.

[0096] 7. ROS-responsive drug release and sustained release

[0097] The drug was encapsulated in a 3500Da dialysis bag and the in vitro release experiment of the drug hydrogel was carried out in PBS buffer containing 4% SDS.

[0098] An ATG group (drug group), an ATG-Se-Se-CS-Gel group (example of the present application), and an ATG-Se-Se-CS-Gel group (example of the present application in an environment with a H2O2 concentration of 0.4%) were set up, with three parallel groups for each group.

[0099] Samples were taken at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 72 hours, and the UV absorbance of the samples was measured at 250 nm using a UV spectrophotometer. The results after deducting the blank were put into the standard curve to obtain the in vitro release dissolution curve of the drug hydrogel. The results are shown in the figure. Figure 8 shown.

[0100] Figure 8 The results showed that the examples of the present application had a significant sustained release effect in the presence of 0.4% H2O2 compared to the drug group without diselenide bonds. Furthermore, compared to the control group without H2O2, they showed significant ROS responsiveness.

[0101] 8. Determination of Intracellular ROS Levels

[0102] Caco-2 cells were selected and the confluent cells were divided into five groups using a six-well plate, namely, the control group, the ATG-Se-Se-CS-Gel group (an example of the present application) in the presence of 150 μM, the ATG group, the CS group, and the vehicle group (only H2O2 was added).

[0103] Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. Add 1 ml of diluted DCFH-DA to each well of a six-well plate. Incubate in a 37°C cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. Detect the results using an enzyme-linked microplate reader at 490 nm. The results are as follows: Figure 9 shown.

[0104] from Figure 9 The results show that the embodiments of the present application can effectively reduce the ROS level of cells.

[0105] 9. DAI score of DSS-induced ulcerative colitis mice

[0106] After the mice were raised for one week to adapt to the environment, they were modeled with 2% DSS for seven days. After that, the drinking water containing 2% DSS was replaced with normal water. The mice were then given drugs by gavage and divided into six groups: control group, 5-ASA group, CS-Gel group, ATG-Se-Se-CS-Gel group (Example of the present application), ATG group, and Model group. The DAI scores of the mice were observed for seven days (the scoring results are shown in Table 2). Figure 10 ).

[0107] Since ulcerative colitis can cause the length of the mouse colon to shorten, generally the more severe the ulcerative colitis is, the shorter the colon will become. By observing the mice in each group, it can be seen that the colon length of the mice administered with the embodiment of the present application is longer than that of the other drug groups ( Figure 11 ). This indicates that the preparation of the present application can effectively inhibit the shortening of the colon length of mice, proving that the preparation of the present application has a significant effect on the treatment of ulcerative colitis.

[0108] Since ulcerative colitis can cause abnormal crypts and neutrophil deposition in mice, HE staining of tissue sections can be used to observe the pathological conditions of the mouse colon tissue and to judge the efficacy of drug treatment. The mice were divided into 6 groups: NC group, ATG-Se-Se-CS-Gel group (an example of the present application), 5-ASA group, ATG group, CS-Gel group, and Model group (wherein the NC group mice were a control group fed normally without any treatment, and the Model group was a control group in which the drinking water containing 2% DSS was replaced with normal drinking water after 7 days of DSS modeling). The results are shown in the figure below. Figure 12 As shown, the pathological condition of the tissue sections of the mice administered with the preparation of the present application was significantly better than that of the other groups.

[0109] 10. Expression of Occludin-1

[0110] The expression of tight junction protein Occludin-1 decreases with the aggravation of ulcerative colitis inflammation. Figure 13 ), it can be seen that the preparation of the present application has a significant therapeutic effect.

[0111] The technical solution of the present application has been described in detail above through specific embodiments. In the above embodiments, the description of each embodiment has its own focus. For parts not described in detail in one embodiment, please refer to the relevant description of other embodiments.

[0112] Those skilled in the art may modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of this application.

Claims

1. A preparation for treating colon inflammation, characterized in that: include: Arctigenin; Chitosan; A cross-linking agent, wherein the cross-linking agent is sodium alginate or genipin; In the preparation, arctigenin and chitosan are connected via a diselenide bond, the substitution ratio of arctigenin to chitosan is (0.25-4):1, and the concentration ratio of the cross-linking agent to the chitosan is 6-9:0.

1.

2. The preparation according to claim 1, characterized in that The concentration ratio of the cross-linking agent to the chitosan is 7-8:0.

1.

3. The preparation according to claim 1, characterized in that The concentration ratio of the cross-linking agent to the chitosan is 7.5:0.

1.

4. The method for preparing the preparation according to any one of claims 1 to 3, wherein: It includes: S1, mixing appropriate amounts of selenomalonic acid and 1-hydroxybenzotriazole and dissolving the mixture in a mixed solution containing dichloromethane and DMSO; S2, adding an appropriate amount of arctigenin to the solution obtained in S1 and reacting for (12-48) hours to obtain a reaction solution; S3, rotary evaporating the reaction solution obtained in S2, adding DMSO to dissolve it, and then sequentially adding appropriate amounts of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), ice-bathing for (1-6) hours, and stirring for activation for (24-72) hours to prepare an active ester; S4, mixing the active ester obtained in S2 with an appropriate amount of chitosan, adding DMSO to dissolve the mixture, and reacting for 12 to 48 hours to obtain the preparation.

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