Application of clofazimine in preparation of medicine for repairing intestinal mucosa or improving intestinal flora

Abstract

The invention provides an application of clofazimine in preparation of a medicine for repairing intestinal mucosa or improving intestinal flora. Experiments prove that clofazimine can effectively repair the abnormal state of intestinal epithelial cells and modify the mechanical barrier of intestinal mucosa; meanwhile, the level of various probiotics is increased, and the level of various harmful bacteria is reduced.

Description

Field of the Invention

[0001] The present application belongs to the field of treatment of digestive tract diseases. Specifically, the present application provides the use of clofazimine in the preparation of drugs for repairing intestinal mucosa or improving intestinal flora. Background Art

[0002] In addition to the function of absorbing nutrients, the intestinal mucosa also has a barrier function to prevent the spread of microorganisms, endotoxins, etc. Specifically, it includes mechanical barriers (rapidly proliferating and tightly arranged cells), chemical barriers (mucin, etc.), immune barriers (secreted antibodies and submucosal lymphoid tissue), and biological barriers (microbial flora). When the intestinal mucosa is damaged due to mechanical trauma, immune disorders, tumors, etc., it may cause a series of problems such as intestinal motility problems, microbial imbalance / translocation, and inflammatory response.

[0003] Clofazimine has significant inhibitory activity against various mycobacteria such as Mycobacterium leprae and Mycobacterium tuberculosis. It is mainly used in the treatment of leprosy in clinical practice. In addition, it has anti-inflammatory and lysosome stabilizing effects, and has certain effects on certain types of lupus erythematosus and certain special skin diseases. There are currently no reports on the use of clofazimine in the treatment of intestinal mucosal-related diseases. Summary of the invention

[0004] On the one hand, the present application provides the use of clofazimine in the preparation of a drug for repairing intestinal mucosa.

[0005] Furthermore, the intestinal mucosa is colon mucosa.

[0006] Furthermore, the repairing of intestinal mucosa is repairing the mechanical barrier of intestinal mucosa.

[0007] Furthermore, the repair of the intestinal mucosal mechanical barrier includes: alleviating swelling of intestinal epithelial cells; maintaining neat arrangement and structural integrity of intestinal microvilli; ensuring normal tight junction gaps and tight connections between intestinal epithelial cells; and reducing intestinal endotoxin translocation.

[0008] The repair of intestinal mucosa may also be repairing the intestinal mucosal biological barrier / improving intestinal flora.

[0009] On the other hand, the present application provides the use of clofazimine in the preparation of a drug for improving intestinal flora.

[0010] Furthermore, the improving intestinal flora includes increasing the level of one or more of Bifidobacteriaceae, Bifidobacterium pseudolongum, Coriobacteriia, Mucispirillum schaedleri, Bacteroides uniformis, Lactobacillus reuteri, Ruminococcus gnavus, Firmicutes, and Actinobacteria in the intestine.

[0011] Furthermore, the improving intestinal flora includes reducing the level of one or more of Clostridium celerecrescens, Ruminococcus flavefaciens, and Proteobacteria in the intestine.

[0012] Furthermore, the drug is in oral or injectable form.

[0013] Furthermore, the drug contains pharmaceutically acceptable excipients.

[0014] The dosage forms available in this application include but are not limited to: tablets, capsules, oral liquids, water injections, powder injections, etc. According to the dosage form prepared / administered, various pharmaceutically acceptable excipients can be selected in the preparation, including but not limited to coating materials, solvents, solubilizers, fillers, disintegrants, binders, stabilizers, antioxidants, pH regulators, osmotic pressure regulators, flavoring agents, taste masking agents, etc. These excipient components can be selected by those skilled in the art according to common knowledge of pharmacy.

[0015] The drug of the present application does not exclude co-administration with other anti-tumor drugs, and these drugs can be co-administered with the drug of the present application or prepared in the same package or preparation.

[0016] The present application found that clofazimine has a significant therapeutic effect on tumors, especially colon cancer, which expands the use of clofazimine and provides a new class of drug options for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the results of transmission electron microscopy examination of colon tissue in the APC model group.

[0018] Figure 2 These are the results of transmission electron microscopy examination of colon tissue in the APC administration group.

[0019] Figure 3 Transmission electron microscopy results of colon tissue in the AOMDSS model group.

[0020] Figure 4Transmission electron microscopy results of colon tissue in the AOMDSS-treated group.

[0021] Figure 5 LPS levels in each group are shown.

[0022] Figure 6 Showing the comparison of the abundance of the main differential bacterial communities between the C57C control group and the C57T drug group.

[0023] Figure 7 The abundance of each bacterial group is shown between the C57C control group and the C57T drug group. DETAILED DESCRIPTION

[0024] Example 1 Basic materials and experimental procedures

[0025] Animal Materials:

[0026] Animals: C57 mice (Speff); Apcmin / + mice (Jicui Yaokang); Gender: male; Age: 6-8 weeks.

[0027] Modeling and processing process:

[0028] Apcmin / + mice:

[0029] After 1 week of adaptive feeding, 20 male mice were divided into two groups, 10 mice in each group.

[0030] Control group: normal feeding, without any intervention or treatment, for 90 days;

[0031] Drug-treated group: The mice were fed normally and given clofazimine (45.5 mg / kg) by gavage once a day for 90 days.

[0032] C57 mice:

[0033] After 1 week of adaptive feeding, 20 male mice were divided into two groups, 10 mice in each group.

[0034] Control group: mice were given a single intraperitoneal injection of 10 mg / kg azoxymethane (AOM). One week after the injection of AOM, the mice were given drinking water containing 2% dextran sodium sulfate (DSS) for 5 days, and then normal drinking water was restored for 16 days, and the cycle was repeated 3 times until the 90th day of the experiment.

[0035] Treatment group: mice were given a single intraperitoneal injection of azoxymethane (AOM) at a dose of 10 mg / kg. One week after the injection of AOM, the mice were given drinking water containing 2% dextran sodium sulfate (DSS) for 5 days, and then normal drinking water was restored for 16 days, with a cycle of 3 times; and at the beginning of the experiment, clofazimine (45.5 mg / kg) was administered orally once a day for 90 days.

[0036] Experimental animals:

[0037] Within 48 hours before dissection, mouse feces were collected and stored at -80 degrees. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, with an injection dose of 0.3 ml / 100 g. The mouse chest cavity was cut open, and blood was collected from the abdominal aorta. Part of the whole blood was taken to separate serum and stored at -80 degrees. The colon tissue was taken out for 4% paraformaldehyde fixation, glutaraldehyde fixation and -80 degrees cryostasis.

[0038] Example 2 Transmission electron microscope detection method

[0039] Equipment and reagents:

[0040] Table 1 Main equipment involved in transmission electron microscopy

[0041]

[0042]

[0043] Table 2 Main reagents involved in transmission electron microscopy

[0044]

[0045] Experimental steps:

[0046] (1) Cell clusters or tissue blocks were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C for 2-4 hours.

[0047] (2) Rinse three times with 0.1 M phosphate buffer (pH 7.4), 15 minutes each time.

[0048] (3) Fixation with 1% osmium acid in 0.1 M phosphate buffer (pH 7.4) at room temperature (20°C) for 2 hours.

[0049] (4) Rinse three times with 0.1 M phosphate buffer (pH 7.4), 15 minutes each time.

[0050] (5) Dehydration: 50%-70%-80%-90%-95%-100%-100% alcohol ascending dehydration, 15 minutes each time.

[0051] (6) Infiltration: Acetone: 812 embedding agent = 1:1 mixed solution infiltration overnight, pure 812 embedding agent infiltration overnight. (7) Embedding: Polymerization at 60 degrees for 48 hours.

[0052] (8) Sectioning: 60-80 nm ultra-thin sections.

[0053] (9) Staining: Uranium-lead double staining (2% saturated aqueous solution of uranyl acetate, lead citrate, each staining for 15 min). The sections were dried at room temperature overnight.

[0054] (10) Electron microscopy observation.

[0055] Example 3 ELISA method for detecting LPS in mouse peripheral blood

[0056] Equipment and reagents:

[0057] LPS ELISA kit (commercial kit).

[0058] Mouse anticoagulated peripheral blood samples (collected from tail vein or cardiac puncture).

[0059] ELISA plates (96-well, flat bottom).

[0060] Sample diluent (provided in the kit).

[0061] Standard diluent.

[0062] Washing solution (PBS or washing solution provided with the kit).

[0063] Chromogenic substrate (TMB) and stop solution.

[0064] Depyrogenate consumables (e.g. microcentrifuge tubes, pipette tips).

[0065] Micropipette.

[0066] ELISA reader (detection wavelength 450nm).

[0067] 37℃ constant temperature incubator.

[0068] Centrifuge.

[0069] Experimental steps:

[0070] Peripheral blood collection:

[0071] Use anticoagulant tubes (EDTA or heparin) to collect peripheral blood from mice to avoid blood coagulation.

[0072] The blood samples were centrifuged at 4°C for 10 minutes (3000 rpm), and the plasma (or serum) was separated and transferred into pyrogen-free EP tubes.

[0073] Processing of plasma or serum:

[0074] If the LPS content is low, the sample may need to be concentrated (eg, by ultrafiltration) to increase the sensitivity of the assay.

[0075] Plasma or serum can be diluted appropriately (usually 1:2 to 1:10), and the specific dilution factor depends on the kit instructions and sample concentration.

[0076] Prepare the standard curve:

[0077] According to the kit instructions, the LPS standard was diluted step by step using standard diluent to prepare gradient concentrations (such as 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1 ng / mL, etc.).

[0078] The standard curve usually includes 6-8 concentration gradients and a blank control (without LPS).

[0079] Each standard concentration was added to the corresponding wells of the ELISA plate, and each well was repeated at least twice (three times is recommended).

[0080] A standard curve (concentration-absorbance value) was drawn according to the absorbance value of the standard.

[0081] Sample testing:

[0082] Plasma or serum samples (after dilution) were added to the test wells of the ELISA plate according to the instructions, and each well was repeated at least twice.

[0083] According to the kit instructions, add capture antibody (usually pre-coated on the ELISA plate), or directly add enzyme-linked antibody (depending on the kit).

[0084] Place the ELISA plate in a 37°C incubator and incubate for 1-2 hours (the specific time is adjusted according to the kit instructions).

[0085] Wash the plate 3-5 times with washing buffer (PBS or provided buffer) to remove non-specifically bound antibodies or samples.

[0086] If the kit requires a second step to add enzyme conjugate, follow the instructions, incubate again, and wash the plate.

[0087] Add TMB substrate (about 50-100 μL per well), incubate for 10-20 minutes, and observe the color reaction (blue gradually appears).

[0088] After adding the stop solution, the color changes from blue to yellow.

[0089] Use an enzyme-labeled instrument to read the absorbance at a wavelength of 450 nm (630 nm can be used as a reference wavelength).

[0090] Substitute the sample absorbance value into the standard curve equation to calculate the LPS concentration in plasma or serum.

[0091] Example 3 16S sequencing to detect bacterial flora

[0092] Sequencing platform: ILLUMINA NovaSeq 6000 sequencing platform; paired-end sequencing PE150; 16S: 5 regions

[0093] Experimental process:

[0094] Bacterial DNA extraction and amplification: Total DNA was extracted from feces using the TIANamp Stool DNA Kit (TIANGEN, China), detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanodropTM 2000 ultra-micro spectrophotometer (ThermoScientific, USA).

[0095] Polymerase chain reaction (PCR) amplification: PCR amplification of the V3-V4 region (341F-806R) of the 16S rRNA gene of the extracted bacterial DNA was performed, with 3 replicates for each sample. The following reaction system was established: System components Volume / mass DNA template 10ng Upstream primer (5mol / L) 0.8L Downstream primer (5mol / L) 0.8L Phusion high-fidelity DNA polymerase (ThermoScientific, USA) 0.4L dNTPs mxi (2.5mmol / L) 2L PCR buffer (5×) 4L BSA 0.2L ddH2O supplemented to 20L Amplification conditions: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 45s, a total of 30 cycles, 72℃ end extension for 8min. PCR instrument: ABI 9700. PCR amplification products were tested by 2% ethidium bromide agarose gel electrophoresis. DNA products were recovered and purified by gel excision using AxyPrep DNA Gel Recovery Kit (Axygen, USA). Products were quantitatively determined by QuantiFluorTM-ST micro-fluorescent agent (Promega, USA). The amplification primer sequences are as follows:

[0096] R1-AGACGTGTGCTCTTCCGATCTCCGTGTCTCAGTCCCARTG (SEQ ID NO. 1);

[0097] R2-AGACTGTGCTCTTCCGATCTGTATTACCGCGGCTGCTG (SEQ ID NO. 2);

[0098] R3-AGACGTGTGCTCTTCCGATCTCCCGTCAATTCMTTTGAGTT (SEQ ID NO.3);

[0099] R4-AGACGTGTGCTCTTCCGATCTCGTTGCGGGACTTAACCC (SEQ ID NO.4);

[0100] R5-AGACGTGTGCTCTTCCGATCTAAGGCCCGGGAACGTATT (SEQ ID NO.5);

[0101] Library construction and sequencing: Nextera XT DNA Sample Preparation Kit (Illumina, USA) was used to construct the sequencing library. Qubit and Q-PCR were used to quantify the constructed library. After the library was qualified, PE250 double-end sequencing was performed on the library using the Illumina NovaSeq 6000 sequencing platform.

[0102] Sequencing data processing: According to the PCR amplification primer sequence and Barcode sequence, the data of each sample is split from the data downloaded from the machine. After cutting off the primer and Barcode sequence, the Reads of the sample are spliced ​​using FLASH2 (Version 2.2.00). The resulting spliced ​​sequence is the original Tags data (Raw Tags), and high-quality Tags data (Clean Tags) are obtained through strict filtering. Referring to the Tag quality control process of QIIME (Version 2.0), the following operations are performed: a) Tags truncation: The Raw Tags are truncated from the first low-quality base site with a continuous low quality value (the default quality threshold is ≤20) and the number of bases reaches the set length (the default length value is 3); b) Tag length filtering: The Tags data set obtained after the Tags are truncated further filters out the Tags whose continuous high-quality base length is less than 75% of the Tag length. The Tag sequences obtained after processing are compared and detected with the species annotation database, and the chimera sequences are finally removed to obtain effective data (Effective Tags).

[0103] Data analysis: Qiime2 software was used to cluster OTUs (Operational Taxonomic Units), and further species annotation and abundance analysis were performed; the complexity of the internal microbial composition was investigated: α diversity (Alpha_Diversity); the differences in microbial composition between groups were analyzed: β diversity (Beta_Diversity); functional analysis, correlation analysis and other data were deeply mined.

[0104] Example 4 Experimental results

[0105] The transmission electron microscopy results are as follows Figure 1-4 As shown:

[0106] APC model group: Intestinal epithelial cells were severely swollen as a whole, the cytoplasmic electron density was unevenly distributed, and some cytoplasms were degraded. Mitochondria in the cytoplasm were severely swollen, cristae were degraded, and the whole was vacuolated. The endoplasmic reticulum was severely swollen and expanded, and the whole was degranulated. The intestinal microvilli were generally degraded and the structure collapsed. The gaps between the tight junctions between cells were large and the connections were not tight.

[0107] APC administration group: Intestinal epithelial cells were slightly swollen overall, cytoplasmic electron density was evenly distributed, cytoplasm was normal without degradation, and there were no vacuoles. Mitochondrial swelling and cristae degradation in the cytoplasm were alleviated, the overall swelling was slight, the endoplasmic reticulum was slightly swollen and expanded, the overall degranulation phenomenon was alleviated, the intestinal microvilli were neatly arranged, the structure was intact without collapse. The tight junction gaps between cells were normal and the connection was tight.

[0108] AOMDSS modeling group: The intestinal epithelial cells were severely swollen as a whole, the cytoplasmic electron density was unevenly distributed, and some cytoplasms were degraded. The mitochondria in the cytoplasm were severely swollen, the cristae were degraded, and the whole was vacuolated. The endoplasmic reticulum was severely swollen and expanded, and the whole was degranulated. The intestinal microvilli were generally degraded and the structure collapsed. The gaps between the tight junctions between cells were large and the connections were not tight.

[0109] AOMDSS administration group: The overall swelling of intestinal epithelial cells was relieved and basically normal, the cytoplasmic electron density was evenly distributed, the cytoplasm was normal without degradation, and there were no vacuoles. The swelling of mitochondria and cristae degradation in the cytoplasm were relieved, the overall swelling was slight, the endoplasmic reticulum was slightly swollen and expanded, the overall degranulation phenomenon was relieved, the intestinal microvilli were neatly arranged, the structure was intact and there was no collapse. The tight junction gaps between cells were normal and the connection was tight.

[0110] LPS test results Figure 2 As shown:

[0111] The LPS levels in the two drug-treated groups were significantly lower than those in the non-drug treated group, indicating that drug-treated groups alleviated the damage to intestinal barrier function.

[0112] The results of bacterial flora testing are as follows Figure 6 , 7 As shown:

[0113] Elevated flora: Bifidobacteriaceae, Coriobacteriia, Mucispirillum schaedleri, Bacteroides uniformis, Lactobacillus reuteri, Bifidobacterium pseudolongum, Ruminococcus gnavus, Firmicutes, Actinobacteria;

[0114] Descending: Clostridium celerecrescens, Bacteroides acidifaciens, Ruminococcus flavefaciens, TM7, Bacteroidetes, Proteobacteria.

[0115] Bacteria that are enhanced by drug effects and their immunomodulatory functions:

[0116] Bifidobacteriaceae and Bifidobacterium pseudolongum

[0117] Bifidobacterium is known for promoting intestinal health and immune regulation, and its metabolites (such as short-chain fatty acids) help maintain the intestinal epithelial barrier and regulate inflammatory responses. Studies have shown that Bifidobacterium can improve anti-tumor immune responses and enhance the efficacy of PD-1 inhibitors by enhancing the activity of dendritic cells (DCs) and T cells.

[0118] Coriobacteriia

[0119] Coriobacteriia is mainly associated with metabolic functions and may indirectly affect the immune system by regulating bile acid metabolism. Secondary bile acids have complex regulatory effects on immune cell activity and may affect T cell differentiation.

[0120] Mucispirillum schaedleri

[0121] Repairs the intestinal mucosa and maintains its integrity; it is related to intestinal mucosal immunity, can enhance local IgA secretion in the intestine, help control pathogens and improve intestinal immune barrier function.

[0122] Bacteroides uniformis

[0123] Certain subtypes of Bacteroides species help reduce intestinal inflammatory responses and improve the immune microenvironment by regulating the balance between regulatory T cells (Treg) and effector T cells.

[0124] Lactobacillus reuteri

[0125] Lactobacillus often regulates immune inflammatory responses by producing lactic acid and other metabolites, promoting the production of short-chain fatty acids, and has been shown to have the ability to regulate Treg cells and enhance anti-inflammatory responses.

[0126] Ruminococcus gnavus

[0127] This flora is associated with the production of short-chain fatty acids in the intestine, which can regulate immune cell activity through the action of short-chain fatty acids. In addition, Ruminococcus metabolism may affect mucosal immune responses.

[0128] Firmicutes and Actinobacteria (phylum level flora)

[0129] Increased Firmicutes are often associated with the production of short-chain fatty acids (such as butyrate), which have positive effects on tumor immunity. Butyrate can modulate T cell function and reduce chronic inflammation.

[0130] The increase of Actinobacteria may also improve immune balance through the production of secondary metabolites.

[0131] Clostridiumcelerecrescens and Ruminococcus flavefaciens

[0132] These bacteria are usually associated with cellulolysis, but some Clostridium species may produce inflammation-related metabolites (such as secondary bile acids), leading to chronic enteritis and immunosuppression.

[0133] Proteobacteria (Phylum Level)

[0134] Proteobacteria are often opportunistic pathogens, and their reduction may reflect improved intestinal health. A decrease in Proteobacteria is often accompanied by a decrease in inflammatory responses.

Claims

1. Application of clofazimine in the preparation of drugs for repairing intestinal mucosa.

2. The use according to claim 1, wherein the intestinal mucosa is colonic mucosa.

3. The use according to claim 1 or 2, wherein the repair of intestinal mucosa is repairing the mechanical barrier of intestinal mucosa.

4. The use according to claim 3, wherein the repairing of the intestinal mucosal mechanical barrier comprises: Relieve swelling of intestinal epithelial cells; keep intestinal microvilli neatly arranged and structurally intact; Ensure that the tight junction gaps between intestinal epithelial cells are normal and tightly connected; reduce intestinal endotoxin translocation.

5. The use according to claim 1, wherein the repair of intestinal mucosa is repair of the intestinal mucosal biological barrier.

6. Application of clofazimine in the preparation of drugs for improving intestinal flora.

7. The use according to claim 6, wherein improving intestinal flora comprises increasing the level of one or more of Bifidobacteriaceae, Bifidobacterium pseudolongum, Coriobacteriia, Mucispirillumschaedleri, Bacteroides uniformis, Lactobacillus reuteri, Ruminococcus gnavus, Firmicutes, and Actinobacteria in the intestine.

8. The use according to claim 6, wherein improving the intestinal flora comprises reducing the level of one or more of Clostridium celerecrescens, Ruminococcus flavefaciens, and Proteobacteria in the intestine.

9. The use according to any one of claims 1 to 8, wherein the drug is in oral or injectable dosage form.

10. The use according to claim 9, wherein the medicine contains pharmaceutically acceptable excipients.

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