Benzocyclooctyne-polyethylene glycol modified halofuginone as well as preparation method and application thereof

Abstract

Hydrophilic modification is carried out on halofuginone through carboxyl polyethylene glycol with a benzocyclooctyne group to obtain benzocyclooctyne-polyethylene glycol-halofuginone (CPH) with good biocompatibility, and the CPH is used for anti-tissue fibrosis treatment through glycometabolism engineering and orthogonal click chemistry. Compared with halofuginone small molecules, the CPH has negligible cytotoxicity. The combination of CPH and glycometabolism engineering has a good treatment effect on tissue fibrosis.

Description

Technical Field

[0001] The invention relates to the field of biomaterials and medical technology, and in particular to benzocyclooctyne-polyethylene glycol-modified halofuginone with good biosafety, and a preparation method and application thereof. Background Art

[0002] Tissue fibrosis is a disease characterized by excessive deposition of connective tissue components in organs caused by disease-induced factors or tissue damage. Excessive accumulation of extracellular matrix (ECM) proteins usually destroys the physiological structure of tissues and may lead to organ dysfunction. Fibrotic tissue remodeling can affect almost all organ systems. Although the incidence of most individual fibrotic diseases is low, fibrotic tissue reactions are very common in asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, or chronic inflammatory bowel disease. For example, myocardial remodeling in heart failure, epithelial-mesenchymal transition and desmoplastic reactions in tumors, and airway remodeling in COPD and asthma. Common diseases associated with fibrosis include hepatitis virus infection, non-alcoholic fatty liver disease (NAFLD), chronic kidney disease, idiopathic pulmonary fibrosis (IPF), myocardial infarction, systemic sclerosis, pneumoconiosis, and cystic fibrosis. The annual combined incidence of major fibrosis-related diseases is approximately 4968 / 100,000 people, resulting in a huge disease burden. Therefore, tissue fibrosis is increasingly recognized as a major health challenge.

[0003] Halofuginone (HF) was first synthesized in the 1960s as a potential antimalarial drug. It is a derivative of the active ingredient of traditional Chinese medicine, halofuginine, and a competitive prolyl-tRNA synthetase. In recent years, researchers have reported that HF ​​can degrade the immunosuppressive extracellular matrix of pancreatic ductal adenocarcinoma and relieve osteoarthritis by inhibiting the activity of TGFβ. Halofuginone is also an effective pulmonary vasodilator that activates Kv channels and blocks voltage-gated calcium ion channels. Halofuginone also has good anti-inflammatory and tumor cell-killing effects. Although halofuginone has anti-malarial, extracellular matrix degradation, anti-inflammatory and vasodilatory effects, halofuginone itself is very toxic to cells, with an IC50 value of nanograms, so its application in vivo is very limited. It is worth noting that the application of halofuginone in the treatment of tissue fibrosis has not been systematically studied, and more importantly, the mechanism of halofuginone's anti-fibrosis is still unclear. Therefore, it is very important to better utilize the anti-fibrotic effect of fusinone to treat tissue fibrosis while ensuring biosafety. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention synthesized benzocyclooctyne polyethylene glycol-modified halofuginone (CPH), and used polyethylene glycol macromolecules with good biocompatibility to reduce the biological toxicity of halofuginone. Through sugar metabolism engineering, the azide group was expressed in fibrotic tissue, and the click chemistry reaction between the azide group and the cycloalkyne group was used to actively target and increase the therapeutic effect of CPH on fibrosis.

[0005] The purpose of the present invention is to provide a CPH with good biosafety and combine it with sugar metabolism engineering technology to enhance the anti-fibrosis effect of CPH.

[0006] The technical solution of the present invention is as follows:

[0007] A benzocyclooctyne-polyethylene glycol-modified halofuginone having the following structure:

[0008]

[0009] Wherein: n=10-110.

[0010] The polyethylene glycol-modified hawthorne is prepared by a room temperature condensation reaction of carboxyl polyethylene glycol (DBCO-PEG-COOH) with a benzocyclooctyne group and the hydroxyl group of hawthorne.

[0011] The PEG used in the present invention may be PEG500, PEG1000, PEG2000, or PEG5000.

[0012] The halofuginone (HF) has the following structure:

[0013]

[0014] Another object of the present invention is to provide a method for preparing the above-mentioned benzocyclooctyne-polyethylene glycol-modified halofuginone, comprising the following steps:

[0015] Step 1: Dissolve DBCO-PEG-COOH and halofuginone of different molecular weights in an aprotic solvent, and then add appropriate amounts of N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) to react.

[0016] The above method is preferably reacted at room temperature for 36 hours.

[0017] Preferably, the molar ratio of DBCO-PEG-COOH:halofuginone:DIC:DMAP is 1:1.2:1.5:0.5.

[0018] Preferably, the aprotic solvent is selected from N,N-dimethylformamide and / or formamide, preferably N,N-dimethylformamide.

[0019] The above method further comprises the steps of removing the reaction solvent and drying.

[0020] Another object of the present invention is to provide the use of the benzocyclooctyne-polyethylene glycol-modified halofuginone of the present invention in drugs for treating tissue fibrosis.

[0021] The benzocyclooctyne-polyethylene glycol-modified fufukone is used in combination with sugar metabolism engineering, wherein the sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine is subjected to sugar metabolism engineering at the fibrosis site of the patient to express the azide group, and then the benzocyclooctyne-polyethylene glycol-modified fufukone is specifically enriched at the lesion site by a click chemistry reaction between the azide group and the benzocyclooctyne-polyethylene glycol-modified fufukone.

[0022] The sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine has the following structure: A specific example of the present invention is that CPH combined with glucose metabolism engineering can effectively treat liver fibrosis

[0023] Advantages of the present invention:

[0024] (1) The benzocyclooctyne-polyethylene glycol-modified halofuginone of the present invention can be used in combination with sugar metabolism engineering to increase the accumulation and retention time of halofuginone in the lesion site, which can better treat tissue fibrosis.

[0025] (2) The CPH preparation method of the present invention is simple and has good biosafety compared to small molecule halofuginone. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0027] Figure 1 This is the NMR characterization of CPH.

[0028] Figure 2 The cytotoxicity of HF on hepatic stellate cells LX2.

[0029] Figure 3 It is the cytotoxicity of CPH to hepatic stellate cells LX2.

[0030] Figure 4 Fluorescence imaging of LX2 glucose metabolism in hepatic stellate cells.

[0031] Figure 5 It is a combination of CPH and glucose metabolism engineering to treat liver fibrosis. DETAILED DESCRIPTION

[0032] The content of the present invention is further explained below in conjunction with embodiments, but these embodiments do not limit the protection scope of the present invention.

[0033] Example 1: Preparation of benzocyclooctyne-polyethylene glycol-halofuginone (CPH)

[0034] Commercial DBCO-PEG 5000 -COOH (Shanghai Pengshuo Biotechnology Co., Ltd.) and halofuginone were dissolved in a molar ratio of 1:1.2 in non-super-dry N,N-dimethylformamide (DMF), and then appropriate amounts of N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine (DMAP) were added to the reaction solution and stirred overnight at room temperature, wherein DBCO-PEG-COOH: halofuginone: DIC: DMAP = 1:1.2:1.5:0.5. After the reaction was completed, DMF was dried by vacuum rotary evaporator, the solid was dissolved in water, the solution was filtered through a 220um water-soluble filter membrane, and the treated aqueous solution was freeze-dried.

[0035] The structural characterization data of the product are as follows Figure 1 As shown, letter a represents the characteristic peak of DBCO group, and its chemical shift is 7.2-7.8ppm; letter b represents the characteristic peak of polyethylene glycol main chain, and the chemical shift at 3.5ppm is the characteristic peak of repeating unit, and the chemical shift at 4.09ppm is the characteristic peak of methylene near carboxyl group; letter c represents the characteristic peak of halofuginone. By integrating the characteristic peak area, it can be calculated that the connection efficiency of halofuginone is 60%.

[0036] Its structure is as follows:

[0037]

[0038] Example 2: CPH cytotoxicity experiment

[0039] Hepatic stellate cells (LX2) in culture flasks were digested and seeded in 96-well plates at a density of 5000 cells / well. Cells were cultured overnight until the cells adhered to the wall. The next day, different concentrations of CPH and halofuginone (HF) were co-cultured with the cells for 48 hours, and the CPH concentration was based on the equivalent amount of HF (the concentration of HF was 10, 20, 40, 60, 80, 100 nM). After 48 hours, the old culture medium was aspirated and 180 μL of fresh culture medium and 20 μL of MTT solution (5 mg / mL) were added to continue culturing in the incubator for 4 hours. The liquid in each well was aspirated and 150 μL of DMSO was added to dissolve in the dark for 10 minutes. The absorbance of each well at 562 nm was measured using an enzyme marker. The cell survival rate was calculated as follows: Cell survival rate (%) = (absorbance value of the test group - background absorbance value) / (absorbance value of the control group - background absorbance value) × 100%.

[0040] The results are as follows Figure 2 and Figure 3As shown, HF is highly toxic to cells, while the modified CPH can significantly downregulate the toxicity of HF and has good biocompatibility.

[0041] Example 3: Glycolytic Engineering to Express Azide on the Cell Surface

[0042] Hepatic stellate cells (LX2) were digested and cultured at 1×10 4 The density of 1000 μg / dish was inoculated in a small dish with a diameter of 35 mm and a glass slide. The cells were cultured in 2 mL DMEM medium overnight, and then 25 μM Ac4ManNAz was added to continue culturing for 48 hours. The cells were washed three times with PBS, 2 mL fresh DMEM medium was added, and then 5 μM DBCO-Cy5 was added and cultured in an incubator for 1 hour. After the culture, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature in the dark for 10 minutes, washed three times with PBS, and the nuclei were stained with DAPI at room temperature in the dark for 10 minutes, washed three times with PBS again, and the cells were observed with a laser confocal microscope under a 63x oil lens.

[0043] The results are as follows Figure 4 As shown, sugar metabolism engineering can successfully express azide groups on the surface of LX2 cells.

[0044] Example 4: CPH and glucose metabolism engineering for the treatment of liver fibrosis

[0045] Liver fibrosis mice were injected with 20 mg / kg Ac4ManNAz or PBS through the tail vein once a day for a total of 3 times. After 3 days, the mice were injected with CPH (0.5 mg / kg HF) or PBS through the tail vein once every two days for a week. Healthy ICR mice were used as negative controls. The mice were killed one week after the end of various administrations, and the main organs were removed and the liver tissue was frozen and sectioned. The liver sections were stained for type I collagen, and the immunofluorescence of the sections was observed using a laser confocal microscope after the staining.

[0046] The results are as follows Figure 5 As shown, CPH combined with glucose metabolism engineering can effectively treat liver fibrosis.

Claims

1. A benzocyclooctyne-polyethylene glycol-modified halofuginone, characterized in that It has the following structure: Wherein: n = 10-110.

2. A method for preparing the benzocyclooctyne-polyethylene glycol-modified halofuginone according to claim 1, characterized in that: It is prepared by condensation reaction of carboxyl polyethylene glycol DBCO-PEG-COOH with benzocyclooctyne group and hydroxyl group of halofuginone.

3. The preparation method according to claim 2, characterized in that The product is prepared by dissolving DBCO-PEG-COOH and halofuginone in an aprotic solvent, adding N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine to undergo condensation reaction.

4. The preparation method according to claim 3, characterized in that The reaction temperature was room temperature and the reaction time was 36 hours.

5. The preparation method according to claim 3, characterized in that The aprotic solvent is selected from N,N-dimethylformamide and / or formamide.

6. The preparation method according to claim 3, characterized in that The molar ratio of DBCO-PEG-COOH: halofuginone: N,N'-diisopropylcarbodiimide: 4-dimethylaminopyridine is = 1:1.2:1.5:0.

5.

7. The preparation method according to claim 3, characterized in that PEG in DBCO-PEG-COOH is PEG500, PEG1000, PEG2000 or PEG5000.

8. Use of the benzocyclooctyne-polyethylene glycol-modified halofuginone according to claim 1 in the preparation of a drug for treating tissue fibrosis.

9. The use according to claim 8, characterized in that The benzocyclooctyne-polyethylene glycol-modified fufukone is used in combination with sugar metabolism engineering, wherein the sugar metabolism precursor tetraacetyl-N-azidoacetylmannosamine is subjected to sugar metabolism engineering at the fibrosis site of the patient to express the azide group, and then the benzocyclooctyne-polyethylene glycol-modified fufukone is specifically enriched at the lesion site by a click chemistry reaction between the azide group and the benzocyclooctyne-polyethylene glycol-modified fufukone.

10. The use according to claim 8, characterized in that The tissue fibrosis is liver fibrosis.

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