Colchicine preparation and application thereof in preparation of medicine for treating organ fibrosis diseases

Abstract

The invention discloses a colchicine preparation and application of the colchicine preparation in preparation of a medicine for treating organ fibrosis diseases, and belongs to the technical field of biological medicines. The colchicine preparation is prepared by taking phospholipid and cholesterol as carriers and loading colchicine, chitosan and interferon-gamma in sequence. According to the preparation, the administration dosage of colchicine is reduced, the bioavailability of colchicine is improved, and the driving effect of inflammation on fibrosis is effectively reduced in cooperation with chitosan; the defects that interferon-gamma is poor in transmembrane permeability and is easily inactivated by enzymolysis in gastrointestinal tracts and the like are overcome, and the stability and the efficiency of the interferon-gamma are remarkably improved. According to the preparation, the immune microenvironment of a focus part is remodeled, and the medicine entrapped in the preparation is helped to play an optimal synergistic effect through a multi-way and multi-target intervention effect; and the pegylated lipidosome can improve the pharmacokinetics of the drug and enhance the delivery efficiency, thereby providing an innovative and prospective solution for the treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a colchicine preparation and application thereof in the preparation of medicines for treating organ fibrosis diseases. Background Art

[0002] Fibrosis is a common pathological feature of many chronic diseases, which manifests as excessive proliferation of connective tissue in diseased organs (such as liver, lungs, kidneys, heart, etc.). As one of the most common types of organ fibrosis in the world, liver fibrosis plays an important role in the study of organ fibrosis. Liver fibrosis is the body's response to liver damage. Alcoholism, viruses, microbial infections, etc. can cause liver damage and lead to liver fibrosis. If no intervention is taken, liver fibrosis can develop into cirrhosis or even the most serious hepatocellular carcinoma. At present, the development of specific drugs for liver fibrosis is still relatively limited. Existing treatment strategies mainly focus on controlling the primary diseases that induce liver fibrosis (such as viral hepatitis, alcoholic liver disease, etc.), but direct intervention methods for liver fibrosis itself still lack effective drug support. Therefore, there is an urgent need to develop a new drug for the treatment of liver fibrosis.

[0003] When liver fibrosis begins, the microenvironment of the lesion site also changes. Hepatic stellate cells have always been considered the main target cells for the treatment of liver fibrosis. Under the action of multiple pathogenic factors, they can be activated into liver myofibroblasts and become the main source of excess extracellular matrix (ECM) in the lesion site of liver fibrosis. However, in recent years, due to the complex pathogenesis of liver fibrosis, researchers have found that the use of drugs with only a single target or target cell has great limitations, and the treatment effect is unsatisfactory. The imbalance of the liver immune microenvironment composed of hepatic stellate cells and abundant natural immune cells in the liver has become an emerging research hotspot for liver fibrosis. People no longer limit the therapeutic targets to hepatic stellate cells, but begin to pay attention to the complex immune microenvironment when liver fibrosis occurs. The research direction mainly revolves around the interaction between liver macrophages, natural killer cells (NKCells) and neutrophils and hepatic stellate cells. Therefore, the development of a new drug that can simultaneously regulate the molecular mechanisms of multiple cells in the lesion site of liver fibrosis, reshape the immune microenvironment of the lesion site, and help anti-fibrosis drugs to achieve the best therapeutic effect is a major problem that needs to be solved urgently.

[0004] Colchicine (COL), also known as colchicine, has the molecular formula: C 22 H 25 NO6, the structural formula is: .

[0005] Colchicine is a well-known ancient medicine with anti-inflammatory, anti-fibrotic, and cell cycle regulating effects. It is often used in clinical practice to treat gout, fibrosis, malignant tumors and other diseases. The main mechanisms of colchicine in treating liver fibrosis are: (1) inhibiting the release of cytokines such as interleukin-1β (IL-1β) and transforming growth factor β1 (TGF-β1) by liver macrophages, reducing the activation of hepatic stellate cells; (2) as an immunomodulator, colchicine can inhibit the expression of nuclear factor κB (NF-κB) and extracellular regulated protein kinase 1 / 2 (ERK1 / 2), participate in the regulation of macrophage polarization, and block the release of proinflammatory cytokines, thereby inhibiting the progression of liver fibrosis; (3) enhancing collagenase activity and promoting collagen decomposition; (4) regulating the hepatic stellate cell cycle and promoting the apoptosis of activated hepatic stellate cells. Despite its wide range of efficacy, its therapeutic disadvantages also make it extremely difficult to use. The first problem to be faced is the extremely narrow medication window. Its effective concentration is very close to the toxic concentration, so it is particularly difficult to control the dosage. Secondly, as an oral drug, its half-life is very short, about 20-60 minutes, so during the treatment process, patients often need to take the drug orally multiple times a day, which can cause a series of side effects, mainly including gastrointestinal symptoms such as abdominal pain and diarrhea. In severe cases, it can also cause poisoning or even shock, with a high risk factor and low patient compliance. Existing studies have shown that nano-delivery of colchicine can reduce its toxic side effects to a certain extent. For example, the colchicine calcium carbonate nanoparticles and colchicine mesoporous silica nanoparticles that have been developed so far are generally larger than 100 nm in size, making it difficult for them to pass through the narrow pores of liver sinusoidal endothelial cells (LSECs) to reach the perisinusoidal space (Disse space) where hepatic stellate cells are located to exert anti-fibrotic effects. In addition, inorganic nanomaterials have certain toxicity, which is inconsistent with the original intention of reducing the toxic side effects of colchicine. Therefore, there is an urgent need to find a way to synergize colchicine, reduce toxicity and improve bioavailability so that it can achieve the best therapeutic effect. Summary of the invention

[0006] The purpose of the present invention is to provide a colchicine preparation and its use in the preparation of drugs for treating organ fibrosis diseases, so as to solve the problems existing in the above-mentioned prior art. By preparing a colchicine preparation that simultaneously encapsulates colchicine, chitosan and interferon-γ, and verifying that it can reverse liver fibrosis through in vivo and in vitro experiments, a new drug and direction are provided for the treatment of liver fibrosis.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a colchicine preparation, which is a nano drug combined preparation which uses phospholipid and cholesterol as carriers and simultaneously loads colchicine, chitosan and interferon-γ.

[0009] The present invention also provides a method for preparing a colchicine preparation, comprising the steps of using phospholipid and cholesterol as carriers to simultaneously load colchicine, chitosan and interferon-γ.

[0010] Optionally, the preparation method comprises the following steps:

[0011] Using phospholipid and cholesterol as carriers, colchicine is loaded on the carriers to synthesize a nano preparation containing colchicine;

[0012] The colchicine-encapsulated nanoformulation is mixed with chitosan for reaction to synthesize the colchicine-encapsulated nanoformulation;

[0013] The colchicine-chitosan-encapsulated nano preparation is mixed with interferon-γ for reaction to synthesize a colchicine-chitosan-interferon-γ-encapsulated nano drug combination preparation, namely, the colchicine preparation.

[0014] Optionally, the method for loading colchicine on the carrier includes any one of (1) or (2):

[0015] (1) dissolving phospholipids and cholesterol in an organic solvent to form a thin film, and then mixing an aqueous medium and a surfactant with the thin film for hydration; subjecting the hydrated system to ultrasonic treatment in an ice bath to obtain a suspension solution, and then mixing and incubating with an aqueous solution of colchicine after dialysis, extruding through a membrane, and purifying by dialysis to obtain a nanoformulation containing colchicine;

[0016] (2) Dissolve phospholipids, cholesterol and surfactant in an organic solvent, and then mix and stir with the colchicine aqueous solution. After the organic solvent in the mixed system is completely evaporated, the mixed system is subjected to ice bath ultrasonic treatment, and then subjected to membrane extrusion and dialysis purification to obtain a nano-preparation containing colchicine.

[0017] In the above scheme, the organic solvent is one of chloroform, dimethyl sulfoxide, anhydrous ethanol, methanol, isopropane, and tert-butanol, or any combination of several of them.

[0018] Optionally, in (1), the ratio of the phospholipid, cholesterol, aqueous medium, surfactant and colchicine aqueous solution is (10-30) mg: (3-5) mg: (0.1-2) mL: (0.005-5) mg: (0.1-2) mL; and / or the mixed incubation conditions are: incubation at 25-65°C for 10-30 min.

[0019] Optionally, in (2), the ratio of the phospholipid, cholesterol, surfactant and colchicine aqueous solution is (10-30) mg: (3-5) mg: (0.005-5) mg: (0.1-2) mL; and / or the conditions of the ice bath ultrasonic treatment are: 100-300W ultrasonic treatment for 5 min under ice bath conditions.

[0020] Optionally, the phospholipid includes at least one of soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine; but the phospholipid of the present invention is not limited to the above types, and can also be one or more of egg yolk lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoyl lecithin, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol or distearoylphosphatidylglycerol, and can also be one or more of all the phospholipids listed above.

[0021] The surfactant includes at least one of Tween 80, poloxamer and Span 20; however, the surfactant of the present invention is not limited to the above types, and can also be one or more of cetyltrimethylammonium bromide, sodium lauryl sulfate, glycerol, sodium lauroyl glutamate and sodium stearate, and can also be one or more of all the above-mentioned surfactants.

[0022] The aqueous medium includes at least one of distilled water, sodium citrate aqueous solution and ammonium sulfate aqueous solution; however, the aqueous medium of the present invention is not limited thereto, and can also be a combination of distilled water and other weak acids, weak bases or their salts.

[0023] Optionally, the conditions for the mixing reaction of the colchicine-encapsulated nanoformulation and chitosan are: after mixing the colchicine-encapsulated nanoformulation with the chitosan solution, magnetic stirring is performed at a speed of 200 rpm for 1-24 hours;

[0024] Calculated based on the colchicine content in the colchicine-encapsulated nanoformulation and the chitosan content in the chitosan solution, the mass ratio of the colchicine to the chitosan is (0.1-5) mg: (0.005-1) mg.

[0025] Optionally, the conditions for the mixed reaction of the colchicine-chitosan-loaded nanoformulation and interferon-γ are as follows: after the colchicine-chitosan-loaded nanoformulation is mixed with the interferon-γ aqueous solution, incubate at 4° C. and 100 rpm for 4-12 hours;

[0026] Calculated based on the colchicine content in the colchicine-chitosan-encapsulated nanoformulation and the interferon-γ content in the interferon-γ aqueous solution, the mass ratio of colchicine to interferon-γ is (0.1-5) mg: (0.005-1) mg.

[0027] The present invention also provides the use of the colchicine preparation in any of the following:

[0028] (1) Application in the preparation of drugs for treating organ fibrosis;

[0029] (2) Application in the preparation of drugs for reversing organ fibrosis;

[0030] Wherein, the organ fibrosis includes liver fibrosis.

[0031] The present invention discloses the following technical effects:

[0032] (1) Remodeling the immune microenvironment and regulating the activation state of hepatic stellate cells: The colchicine preparation prepared by the present invention is a nano-drug combination preparation that uses phospholipids and cholesterol as carriers to simultaneously load colchicine, chitosan and interferon-γ. It uses colchicine, chitosan and interferon-γ as anti-fibrotic active ingredients to inhibit the activation of hepatic stellate cells. The combination of multiple components jointly regulates the immune microenvironment of the liver fibrosis lesion site through multi-pathway and multi-target intervention, thereby exerting the best synergistic anti-fibrosis therapeutic effect.

[0033] (2) Regulating macrophage polarization and maintaining immune system homeostasis: Colchicine, as an immunomodulator, works together with chitosan encapsulated in nanoformulations to regulate the polarization balance of macrophages in the early stages of liver fibrosis, achieving the goal of synergistic anti-liver fibrosis.

[0034] (3) Reduce the number of hepatic myofibroblasts and delay the progression of liver fibrosis: During the advanced stage of liver fibrosis, interferon-γ inhibits the proliferation and activation of hepatic stellate cells into hepatic myofibroblasts. Colchicine, as an anti-mitotic agent, can effectively regulate the cell cycle of hepatic myofibroblasts and promote their apoptosis. The two synergistically reduce the number of hepatic myofibroblasts and significantly enhance the inhibitory effect on liver fibrosis.

[0035] (4) Inhibit collagen deposition in liver lesions: Colchicine and interferon-γ work synergistically to promote collagen decomposition, reduce collagen deposition, and reverse liver fibrosis.

[0036] (5) Preparation of small-particle nano-platforms with high encapsulation efficiency and good stability to improve targeting efficiency: The colchicine preparation provided by the present invention has a novel synthesis method and an encapsulation efficiency of colchicine of more than 80%. While loading small molecule drugs (colchicine), polymer compounds (chitosan) and proteins (interferon-γ) at the same time, the particle size is only about 80nm. The ultra-small size advantage enables it to pass through the narrow pores of liver sinusoidal endothelial cells smoothly to reach the lesion site and exert its therapeutic effect.

[0037] (6) High biosafety and great potential for clinical transformation: The ingredients used in the colchicine preparation provided by the present invention are safe, which overcomes the problem of large toxic and side effects of using a single anti-fibrosis drug. In addition, the PEGylated liposomes greatly prolong the drug's circulation time in the body. Experimental data show that when the dosage and frequency of administration are only one-tenth of the original drug, it exhibits a more significant effect in reversing liver fibrosis.

[0038] (7) The present invention reduces the dosage of colchicine while improving its bioavailability, and synergizes with chitosan to effectively reduce the driving effect of inflammation on fibrosis; it overcomes the defects of interferon-γ such as poor transmembrane permeability and easy enzymatic inactivation in the gastrointestinal tract, significantly improves its stability and efficacy, and provides an innovative and forward-looking solution for the treatment of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The L prepared by the present invention COL-CS-IFN Transmission electron microscopy images of nanoformulations;

[0041] Figure 2 The L prepared by the present invention COL , L COL-CS , L COL-CS-IFN Particle size comparison of nanoparticles; A: L COL , B:L COL-CS , C:L COL-CS-IFN ;

[0042] Figure 3 The L prepared by the present invention COL , L COL-CS , L COL-CS-IFN Potential comparison diagram of nanoformulation and interferon-γ aqueous solution;

[0043] Figure 4 The L prepared by the present invention COL , L COL-CS , L COL-CS-IFN Comparison of polymer dispersibility index of nanoformulations;

[0044] Figure 5 To monitor the L prepared by the present invention COL-CS-IFN Long-term stability of particle size and polymer dispersibility index of nanoformulations; A: particle size, B: polymer dispersibility index;

[0045] Figure 6 For the scratch test, L COL , L COL-CS-IFN Effects of nanoparticles on the migration of LX-2 cells; A: Microscope observation images, B: Data statistical results;

[0046] Figure 7 To investigate L COL , L COL-CS-IFN Toxicity of nanoparticles to LX-2 cells; A: L COL , B:L COL-CS-IFN ;

[0047] Figure 8 Flow cytometry was used to investigate L COL , L COL-CS-IFN Effects of nanoformulations on apoptosis of LX-2 cells; A: flow cytometry images, B: statistical results;

[0048] Fig. 9 Real-time fluorescence quantitative polymerase chain reaction (qPCR) assay to detect L COL , L COL-CS-IFN Effects of nanoformulations on the relative mRNA expression of IL-1β, TGF-β1, NF-κB, and ERK1 / 2 produced by macrophages; A: relative mRNA expression of IL-1β, B: relative mRNA expression of TGF-β1, C: relative mRNA expression of NF-κB, D: relative mRNA expression of ERK1 / 2;

[0049] Fig.10 This is a comparison of the weight changes of mice in different treatment groups in an in vivo liver fibrosis animal experiment over 8 weeks;

[0050] Fig.11 This is the appearance of liver tissues of mice in different treatment groups in the in vivo liver fibrosis animal experiment;

[0051] Fig.12 This is a comparison chart of liver indexes of mice in different treatment groups in an in vivo liver fibrosis animal experiment;

[0052] Fig.13 These are the H&E, Masson's, and Sirius red staining images of the livers of mice in different treatment groups in the in vivo liver fibrosis animal experiment;

[0053] Fig.14 This is the immunohistochemical staining of COL1A1 in the liver of mice in different treatment groups in the in vivo liver fibrosis animal experiment;

[0054] Fig.15The mRNA expression levels of COL1A1 and COL3A1 in the liver of mice in different treatment groups were detected by qPCR experiment; A: relative mRNA expression of COL1A1, B: relative mRNA expression of COL3A1;

[0055] Fig.16 These are the shear wave ultrasound elastic imaging images of the liver of mice in different treatment groups in the in vivo liver fibrosis animal experiment; A: shear wave ultrasound elastic imaging image, B: data statistical results;

[0056] Fig.17 Healthy mice were intravenously injected with saline and L COL-CS-IFN H&E staining of the heart, liver, spleen, lung and kidney organs of the nanoformulation. DETAILED DESCRIPTION

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0059] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0060] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0061] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0062] COL: Colchicine;

[0063] CS: Chitosan;

[0064] IFN-γ: interferon-gamma.

[0065] Example 1 A method for preparing a colchicine preparation

[0066] (1) Use distilled water in the aqueous medium to prepare 0.3 mL of 2 mg / mL COL aqueous solution, 0.3 mL of 100 mM / L ammonium sulfate aqueous solution, 0.2 mL of 0.5 mg / mL CS solution (the solvent is 0.5% weak acid solution), and 0.2 mL of 500 μg / mL IFN-γ aqueous solution;

[0067] (2) Dissolve 18.2 mg of soybean lecithin, 1.8 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 3.5 mg of cholesterol in 1 mL of chloroform, and perform rotary evaporation to form a film. The obtained film is vacuum dried overnight for later use;

[0068] (3) adding the ammonium sulfate aqueous solution and 30 μg of Tween 80 prepared in step (1) to the film obtained in step (2), and shaking the film ultrasonically in a water bath at 45°C for 5 min to hydrate the film;

[0069] (4) The hydrated system obtained in step (3) was subjected to ultrasonic treatment at 100W for 5 min under ice bath conditions to obtain a suspension solution, which was dialyzed for 4 h at 4°C to remove excess ammonium sulfate in the system for later use;

[0070] (5) The COL aqueous solution prepared in step (1) was added to the dialyzed suspension obtained in step (4), and the mixture was incubated at 55° C. for 30 min. After cooling to room temperature, the mixture was subjected to film extrusion and dialyzed to obtain a lipid nanoformulation containing COL. The nanoformulation was named L COL ;

[0071] (6) L COL The lipid nanoformulation was mixed with the CS solution prepared in step (1) and magnetically stirred at 200 rpm for 6 h to obtain the COL-CS nanoformulation, which was named L COL-CS ;

[0072] (7) The IFN-γ aqueous solution prepared in step (1) and the L obtained in step (6) are mixed. COL-CS The nanoformulation was incubated on a shaker at 4°C and 100 rpm for 12 h, and IFN-γ was successfully loaded by electrostatic adsorption. Then, free IFN-γ was removed by ultrafiltration centrifugation at 4°C and 10,000 rpm for 20 min, resulting in a nano-drug combination preparation, namely colchicine preparation, named LCOL-CS-IFN .

[0073] Example 2 A method for preparing a colchicine preparation

[0074] (1) Use distilled water in the aqueous medium to prepare 0.2 mL of 2 mg / mL COL aqueous solution, 0.2 mL of 400 mM / L sodium citrate aqueous solution, 0.4 mL of 0.4 mg / mL CS solution (the solvent is 0.5% weak acid solution), and 0.2 mL of 500 μg / mL IFN-γ aqueous solution;

[0075] (2) Dissolve 18.2 mg of soybean lecithin, 1.8 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 3.5 mg of cholesterol in 1 mL of chloroform, and perform rotary evaporation to form a film. The obtained film is vacuum dried overnight for later use;

[0076] (3) adding the sodium citrate aqueous solution prepared in step (1) and 30 μg of Span 20 to the film obtained in step (2), and shaking the film by ultrasonic in a water bath at 45°C for 5 min to hydrate the film;

[0077] (4) The hydration system prepared in step (3) was subjected to ultrasonic treatment at 200W for 5 min in an ice bath to obtain a suspension solution, which was dialyzed for 4 h at 4°C to remove excess sodium citrate in the system for later use;

[0078] (5) adding the COL aqueous solution prepared in step (1) to the dialyzed suspension obtained in step (4), incubating at 25° C. for 20 min, cooling to room temperature, and purifying by thin film extrusion and dialyzing to obtain a lipid nanoformulation encapsulating COL;

[0079] (6) mixing the COL-loaded lipid nanoformulation with the CS solution prepared in step (1), and magnetically stirring the mixture at 200 rpm for 4 h to obtain a COL-CS nanoformulation;

[0080] (7) The IFN-γ aqueous solution prepared in step (1) and the COL-CS nanoformulation obtained in step (6) were incubated on a shaker at 4°C and 100 rpm for 10 h, IFN-γ was successfully loaded by electrostatic adsorption, and then ultrafiltration centrifugation was performed at 4°C and 12000 rpm for 20 min to remove free IFN-γ, thereby obtaining a nano-drug combination preparation, namely, colchicine preparation.

[0081] Example 3 A method for preparing a colchicine preparation

[0082] (1) Use distilled water in the aqueous medium to prepare 0.2 mL of 5 mg / mL COL aqueous solution, 0.2 mL of 300 mM / L ammonium sulfate aqueous solution, 0.4 mL of 0.4 mg / mL CS solution (the solvent is 0.5% weak acid solution), and 0.2 mL of 800 μg / mL IFN-γ aqueous solution;

[0083] (2) Dissolve 16.4 mg of soybean lecithin, 3.6 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 4.5 mg of cholesterol in 1 mL of chloroform, and perform rotary evaporation to form a film. The obtained film is vacuum dried overnight for later use;

[0084] (3) adding the ammonium sulfate aqueous solution and 1 mg of poloxamer prepared in step (1) to the film obtained in step (2), and ultrasonically shaking the film in a water bath at 45° C. for 8 min to hydrate the film;

[0085] (4) The hydration system obtained in step (3) was subjected to ultrasonic treatment at 300W for 5 min under ice bath conditions to obtain a suspension solution, which was dialyzed at 4°C for 4 h to remove excess ammonium sulfate in the system for later use;

[0086] (5) adding the COL aqueous solution obtained in step (1) to the dialyzed suspension obtained in step (4), incubating at 60° C. for 20 min, cooling to room temperature, and performing thin film extrusion and dialysis purification to obtain a lipid nanoformulation encapsulating COL;

[0087] (6) mixing the COL-loaded lipid nanoformulation with the CS solution prepared in step (1), and magnetically stirring the mixture at 200 rpm for 8 h to obtain a COL-CS nanoformulation;

[0088] (7) The IFN-γ aqueous solution prepared in step (1) and the COL-CS nanoformulation obtained in step (6) were incubated on a shaker at 4°C and 100 rpm for 4 h, IFN-γ was successfully loaded by electrostatic adsorption, and then ultrafiltration centrifugation was performed at 4°C and 15,000 rpm for 20 min to remove free IFN-γ, thereby obtaining a nano-drug combination preparation, namely, a colchicine preparation.

[0089] Example 4 A method for preparing a colchicine preparation

[0090] (1) Use distilled water in the aqueous medium to prepare 0.2 mL of 1 mg / mL COL aqueous solution, 0.2 mL of 200 mM / L ammonium sulfate aqueous solution, 0.4 mL of 0.2 mg / mL CS solution (the solvent is 0.5% weak acid solution), and 0.2 mL of 500 μg / mL IFN-γ aqueous solution;

[0091] (2) Dissolve 16.4 mg of dipalmitoylphosphatidylcholine, 3.6 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 3.5 mg of cholesterol in 1 mL of chloroform, and perform rotary evaporation to form a film. The obtained film is vacuum dried overnight for later use;

[0092] (3) adding the ammonium sulfate aqueous solution and 30 μg of Tween 80 prepared in step (1) to the film obtained in step (2), and shaking the film in a 45°C water bath with ultrasound for 10 min to hydrate the film;

[0093] (4) The hydrated system obtained in step (3) was subjected to ultrasonic treatment at 100W for 5 min under ice bath conditions to obtain a suspension solution, which was dialyzed for 4 h at 4°C to remove excess ammonium sulfate in the system for later use;

[0094] (5) adding the COL aqueous solution prepared in step (1) to the dialyzed suspension obtained in step (4), incubating at 45° C. for 10 min, cooling to room temperature, and then performing thin film extrusion and dialysis purification to obtain a lipid nanoformulation encapsulating COL;

[0095] (6) mixing the COL-loaded lipid nanoformulation with the CS solution prepared in step (1), and magnetically stirring at 200 rpm for 12 h to obtain a COL-CS nanoformulation;

[0096] (7) The IFN-γ aqueous solution prepared in step (1) and the COL-CS nanoformulation obtained in step (6) were incubated on a shaker at 4°C and 100 rpm for 6 h, IFN-γ was successfully loaded by electrostatic adsorption, and then ultrafiltration centrifugation was performed at 4°C and 10,000 rpm for 20 min to remove free IFN-γ, thereby obtaining a nano-drug combination preparation, namely, a colchicine preparation.

[0097] Example 5 A method for preparing a colchicine preparation

[0098] (1) Use distilled water in the aqueous medium to prepare 0.2 mL of 1 mg / mL COL aqueous solution, 0.2 mL of 200 mM / L ammonium sulfate aqueous solution, 0.4 mL of 0.5 mg / mL CS solution (the solvent is 0.5% weak acid solution), and 0.2 mL of 500 μg / mL IFN-γ aqueous solution;

[0099] (2) Dissolve 13.2 mg of dipalmitoylphosphatidylcholine, 3.3 mg of dipalmitoylphosphatidylethanolamine, 3.5 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, and 4.5 mg of cholesterol in 1 mL of chloroform, and perform rotary evaporation to form a film. The obtained film is vacuum dried overnight for later use;

[0100] (3) adding the ammonium sulfate aqueous solution and 30 μg of Tween 80 prepared in step (1) to the film obtained in step (2), and shaking the film in a 45°C water bath with ultrasound for 10 min to hydrate the film;

[0101] (4) The hydration system obtained in step (3) was subjected to ultrasonic treatment at 300W for 5 min under ice bath conditions to obtain a suspension solution, which was dialyzed at 4°C for 4 h to remove excess ammonium sulfate in the system for later use;

[0102] (5) adding the COL aqueous solution prepared in step (1) to the dialyzed suspension obtained in step (4), incubating at 65° C. for 20 min, cooling to room temperature, and then performing thin film extrusion and dialysis purification to obtain a lipid nanoformulation encapsulating COL;

[0103] (6) mixing the COL-loaded lipid nanoformulation with the CS solution obtained in step (1), and magnetically stirring at 200 rpm for 2 h to obtain a COL-CS nanoformulation;

[0104] (7) The IFN-γ aqueous solution prepared in step (1) and the COL-CS nanoformulation obtained in step (6) were incubated on a shaker at 4°C and 100 rpm for 12 h, IFN-γ was successfully loaded by electrostatic adsorption, and then ultrafiltration centrifugation was performed at 4°C and 10,000 rpm for 20 min to remove free IFN-γ, thereby obtaining a nano-drug combination preparation, namely, a colchicine preparation.

[0105] Example 6 A method for preparing a colchicine preparation

[0106] (1) Use distilled water as the aqueous medium to prepare 0.4 mL of 0.5 mg / mL COL aqueous solution, 0.4 mL of 0.2 mg / mL CS solution (the solvent is a 0.5% weak acid solution), and 0.2 mL of 500 μg / mL IFN-γ aqueous solution;

[0107] (2) Dissolve 16.4 mg of soybean lecithin, 3.6 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 4 mg of cholesterol, and 30 μg of Tween 80 in 50°C pure ethanol, and inject the resulting solution into the COL aqueous solution obtained in step (1) at a uniform speed;

[0108] (3) After the ethanol in the stirring system in step (2) is evaporated and removed, ultrasonic treatment is performed at 200W for 5 minutes under ice bath conditions to obtain a suspension solution, which is then subjected to membrane extrusion and dialyzed for purification to obtain a lipid nanoparticle preparation containing COL;

[0109] (4) mixing the COL-loaded lipid nanoformulation with the CS solution prepared in step (1), and magnetically stirring the mixture at 200 rpm for 2 h to obtain a COL-CS nanoformulation;

[0110] (5) The IFN-γ aqueous solution prepared in step (1) and the COL-CS nanoformulation obtained in step (4) were incubated on a shaker at 4°C and 100 rpm for 12 h, IFN-γ was successfully loaded by electrostatic adsorption, and then ultrafiltration centrifugation was performed at 4°C and 12000 rpm for 20 min to remove free IFN-γ, thereby obtaining a nano-drug combination preparation, namely, colchicine preparation.

[0111] Example 7 Investigation of the microscopic morphology of colchicine preparations

[0112] The colchicine preparation L prepared in Example 1 COL-CS-IFN Add it to the copper grid, dry it, add 1 drop of 2% phosphotungstic acid staining solution to the copper grid, stain it for 30 seconds, absorb the staining solution, dry it, and take a transmission electron microscope (TEM) photo. The results are as follows Figure 1 As shown: L prepared by the present invention COL-CS-IFN The morphology is round and regular, the particle size is uniform, and the average particle size is about 70nm.

[0113] Example 8 Dynamic Light Scattering (DLS) Characterization of Colchicine Preparations

[0114] Calculation of L by high performance liquid chromatography COL-CS-IFN The encapsulation efficiency of colchicine is about 85%. The particle size of L COL , L COL-CS , L COL-CS-IFN The particle size (Size), potential (Zeta), polymer dispersibility index (PDI) of the three nanoformulations and the potential of IFN-γ aqueous solution are shown in the figure. Figure 2-4 As shown, the changes in particle size and potential confirm that L COL-CS-IFN The nanoformulation was successfully prepared. Monitoring L COL-CS-IFN Particle size and polymer dispersibility index of nanoformulations, e.g. Figure 5 As shown, there is no significant change in particle size and polymer dispersibility index within one week, which confirms that the colchicine preparation prepared by the present invention has long-term stability.

[0115] Example 9 Cell scratch assay to investigate the effect of colchicine preparations on the migration of human hepatic stellate cells (LX-2)

[0116] Four LX-2 cell groups were set up: Control group (DMEM complete medium), TGF-β1 group (DMEM complete medium supplemented with TGF-β1), L COL Group (supplemented with TGF-β1 and L COL DMEM complete medium), L COL-CS-IFN Group (supplemented with TGF-β1 and L COL-CS-IFNDMEM complete medium) with TGF-β1 concentration of 10 ng / mL and COL concentration of 30 ng / mL. 5 The cells were seeded into a 6-well plate and placed in a constant temperature incubator for 24 hours. A cell-free scratch was made with a gun tip, and the cells were photographed under a microscope. This was the 0th hour. The plates were then placed in a constant temperature incubator for another 24 hours and photographed again under a microscope. This was the 24th hour. Figure 6 As shown in the analysis results, compared with the TGF-β1 group, L COL-CS-IFN The inhibition of cell migration ability in the group was stronger than that in L COL This suggests that the three active ingredients synergistically inhibited LX-2 cell migration.

[0117] Example 10 MTT method to investigate the effect of colchicine preparation on LX-2 cell proliferation

[0118] The L prepared in Example 1 was investigated respectively. COL and L COL-CS-IFN Effect of nanoformulation on the proliferation activity of LX-2 cells. LX-2 cells growing in the logarithmic phase were taken and TGF-β1 was added to induce activation of LX-2 cells. The cell density was adjusted to 8×10 3 96-well plates were placed in each well and incubated overnight in a constant temperature incubator. COL and L COL-CS-IFN The nanopreparation was administered at the concentration, and the cells were placed in a constant temperature incubator for 24 hours after the drug was added. Then 20 μL of MTT solution (PBS dissolved, concentration of 5 mg / mL) was added to each well, and the cells were cultured for 4 hours under the same culture conditions. The culture medium was aspirated, 150 μL of dimethyl sulfoxide (DMSO) was added and shaken for ten minutes, and the absorbance (A) of each well at 490 nm was measured using an enzyme reader to calculate the cell proliferation rate. The formula for calculating the cell proliferation rate is: (A drug group-A zero adjustment group) / (A control group-A zero adjustment group)×100%.

[0119] The results are as follows Figure 7 As shown, L COL and L COL-CS-IFN Nanoparticles can reduce the proliferation activity of activated LX-2 cells in a concentration-dependent manner. COL-CS-IFN The inhibitory effect of group on cell proliferation was stronger than that of group L. COL Group.

[0120] Example 11 Investigating the effect of colchicine preparation on LX-2 cell cycle

[0121] After the onset of liver fibrosis, hepatic stellate cells activate into hepatic myofibroblasts and become the main source of excessive collagen fibers, so their cell cycle is closely related to the process of fibrosis. LX-2 cells were activated by TGF-β1, and the same grouping as the cell scratch experiment was set. After staining with an apoptosis kit, the apoptosis of each group was detected by flow cytometry.

[0122] The results are as follows Figure 8 As shown, L COL and L COL-CS-IFN Nanoparticles can promote apoptosis of LX-2 cells. COL-CS-IFN The effect of the nanoformulation in promoting cell apoptosis was more significant, which confirmed the synergistic effect between COL, CS and IFN-γ in jointly regulating the LX-2 cell cycle.

[0123] Example 12 Investigating the Effect of Colchicine Preparations on the Expression Levels of Profibrotic Mediators Produced by Macrophages

[0124] After lipopolysaccharide (LPS) induced macrophages (Raw264.7) for 24 hours, the expression levels of IL-1β, TGF-β1, NF-κB, and ERK1 / 2 increased significantly. These cytokines induce LX-2 cell activation, promote M1 macrophage polarization, and thus promote the process of liver fibrosis. The effects of the colchicine preparation prepared in Example 1 of the present invention on these profibrotic media were investigated. Four Raw264.7 cell groups were set up: Control group (DMEM complete medium), LPS group (DMEM complete medium with LPS added), L COL Group (add LPS and L COL DMEM complete medium), L COL-CS-IFN Group (add LPS and L COL-CS-IFN DMEM complete medium) with LPS concentration of 0.1 μg / mL and COL concentration of 30 ng / mL. COL , L COL-CS-IFN After the nanoformulations were incubated with LPS-induced Raw264.7 cells for 24 h, real-time fluorescence quantitative polymerase chain reaction (qPCR) experiments were used to detect the mRNA expression levels of IL-1β, TGF-β1, NF-κB, and ERK1 / 2 in cells of different treatment groups.

[0125] The results are as follows Fig. 9 As shown, L COL-CS-IFN The down-regulation effect of the four profibrotic mediators in group A was significantly better than that in group B. COL group, indicating that COL and CS can synergistically inhibit macrophage activity, reduce LX-2 cell activation, regulate the polarization balance of macrophages, improve the immune microenvironment at the lesion site, and alleviate the severity of liver fibrosis.

[0126] Example 13 Establishment and treatment of mouse liver fibrosis model

[0127] C57BL / 6 mice were selected to establish a liver fibrosis model, and four animal experimental groups were set up: healthy group (Healthy), carbon tetrachloride group (CCl4), L COL Group, L COL-CS-IFN All groups except the Healthy group were intraperitoneally injected with CCl4 olive oil suspension (2.5 μL / g, CCl4: olive oil = 1:4 (V:V)) twice a week for eight weeks. COL Group, L COL-CS-IFN All groups started treatment from the 5th week after modeling, and the corresponding nanoformulation was injected into the rat tail vein twice a week, with a COL dosage of 0.1 mg / kg each time. The body weight of mice in each group was recorded every week. Fig.10 The results show that the weight of mice in each treatment group increased to a certain extent. COL-CS-IFN The weight of mice in group A was close to that of mice in group B.

[0128] After the treatment, blood was collected from the mouse orbits, and the serum was separated and frozen in a -80°C refrigerator for subsequent experiments. All mice were killed and the heart, liver, spleen, lung, and kidney organs were collected. The appearance of the liver of each group of mice was recorded and the liver index of the mice was calculated. The results are as follows Fig.11 and Fig.12 As shown in the figure, the liver index calculation formula is: liver index (%) = mouse liver weight / mouse body weight × 100%. COL-CS-IFN The increase in body weight of mice in the Healthy group, the decrease in liver index, and the smooth and soft appearance of the liver close to that of mice in the Healthy group all strongly confirmed the significant anti-fibrosis therapeutic effect of the colchicine preparation prepared by the present invention.

[0129] Example 14 H&E, Masson's, and Sirius Red staining of mouse liver tissue sections

[0130] Mouse liver tissue was fixed, dehydrated, embedded, and sliced, and then stained with H&E, Masson, and Sirius red. The results are as follows: Fig.13 The results of liver staining of mice in each group are shown. First, the results of H&E staining were analyzed. Compared with the CCl4 group, the liver fibrosis of the treatment group was alleviated to varying degrees. COL-CS-IFN The effect of the group was the best, close to normal liver tissue, and normal hepatocyte morphology and clear and complete liver lobule structure could be seen. Then, the results of Masson and Sirius red staining were analyzed, and L COL-CS-IFN The collagen fiber deposition in the L COL-CS-IFN It can slowly release anti-fibrotic active ingredients in the body, and the multiple components synergistically enhance the effects, effectively reversing liver fibrosis in mice.

[0131] Example 15 Immunohistochemical staining of COL1A1 in mouse liver tissue sections

[0132] The liver tissue was dewaxed and hydrated. After antigen repair, 3% hydrogen peroxide solution was used to remove endogenous peroxidase, serum was blocked, and incubated with COL1A1-specific primary antibody overnight at 4°C, and the secondary antibody was incubated for 1 hour at room temperature. DAB colorimetric solution was added for staining, and the slides were counterstained with hematoxylin after rinsing with distilled water. Gradient alcohol and xylene were used for dehydration, neutral gum was used for sealing, and the slides were placed under a microscope for photography. The staining results are shown in Figure 2. Fig.14 As shown, L COL-CS-IFN The positive expression of COL1A1 in the treatment group was significantly reduced, and the effect was better than that in L COL This further confirms that L COL-CS-IFN The synergistic effect between the components can effectively improve collagen deposition in liver fibrosis lesions.

[0133] Example 16 qPCR experiment to detect the mRNA expression levels of COL1A1 and COL3A1 in mouse liver tissue

[0134] Literature has shown that COL can enhance the activity of collagenase, and IFN-γ can reduce the expression levels of COL1A1 and COL3A1 mRNA. The two can play a synergistic role in reducing collagen deposition. Liver tissue samples were prepared, and qPCR experiments were performed to detect the mRNA expression levels of COL1A1 and COL3A1. The results are as follows Fig.15 As shown in Figure 2, the expression levels of COL1A1 and COL3A1 were significantly downregulated after treatment, and L COL-CS-IFN The down-regulation effect of group was significantly better than that of L COL This further confirms that L COL-CS-IFN The components of the nanoformulation work synergistically to enhance the therapeutic effect of liver fibrosis.

[0135] Example 17 Shear wave ultrasound elastography to detect the therapeutic effect of colchicine preparation on liver fibrosis

[0136] Shear wave ultrasound elastography is an advanced imaging method for diagnosing liver fibrosis and monitoring its progression. It can non-invasively detect liver stiffness and assess the severity of liver fibrosis. Shear wave ultrasound elastography was performed on the livers of mice in each group, and the treatment effect was evaluated by measuring the liver Young's modulus (E). Fig.16 As shown, we can see that L COL-CS-IFN After treatment, the Young's modulus value was significantly reduced and was very close to that of the liver of normal mice. The experimental results verified the effectiveness of the colchicine preparation prepared by the present invention in reversing liver fibrosis in imaging.

[0137] Example 18 Verification of the biosafety of colchicine preparations

[0138] The safety of a drug is an important factor in its development and application. Examining the biosafety of the colchicine preparation prepared in Example 1 of the present invention is crucial to evaluating its clinical application value. Healthy C57BL / 6 mice were randomly divided into two groups and intravenously injected with physiological saline and colchicine preparation L COL-CS-IFN The drug was administered twice a week for 4 consecutive weeks. 24 hours after the last administration, the mice were killed, and the heart, liver, spleen, lung, and kidney were removed. After fixation with 4% paraformaldehyde, the sections were embedded in paraffin and stained with H&E. The pathological conditions of the mouse organs were observed under an optical microscope. The results are as follows: Fig.17 As shown in the results, compared with the saline group, L COL-CS-IFN There were no obvious lesions and injuries in the organs of the group, indicating that it would not produce obvious organ toxicity.

[0139] The above experiments prove that the colchicine preparation L prepared by the present invention COL-CS-IFN The active ingredients of the drug synergize with each other to reshape the liver's immune microenvironment, significantly reduce collagen deposition in liver tissue, and have excellent biosafety and anti-fibrosis effects.

[0140] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A colchicine preparation, characterized in that, The invention is a nano drug combination preparation which uses phospholipid and cholesterol as carriers and simultaneously loads colchicine, chitosan and interferon-γ.

2. A method for preparing the colchicine preparation as claimed in claim 1, characterized in that: The method comprises the steps of using phospholipid and cholesterol as carriers to simultaneously load colchicine, chitosan and interferon-γ.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: Using phospholipid and cholesterol as carriers, colchicine is loaded on the carriers to synthesize a nano preparation containing colchicine; The colchicine-encapsulated nanoformulation is mixed with chitosan for reaction to synthesize the colchicine-encapsulated nanoformulation; The colchicine-chitosan-encapsulated nano preparation is mixed with interferon-γ for reaction to synthesize a colchicine-chitosan-interferon-γ-encapsulated nano drug combination preparation, namely, the colchicine preparation.

4. The preparation method according to claim 3, characterized in that: The method for loading colchicine on the carrier includes any one of (1) or (2): (1) dissolving phospholipids and cholesterol in an organic solvent to form a thin film, and then mixing an aqueous medium and a surfactant with the thin film for hydration; subjecting the hydrated system to ultrasonic treatment in an ice bath to obtain a suspension solution, and then mixing and incubating with an aqueous solution of colchicine after dialysis, extruding through a membrane, and purifying by dialysis to obtain a nanoformulation containing colchicine; (2) Dissolve phospholipids, cholesterol and surfactant in an organic solvent, and then mix and stir with the colchicine aqueous solution. After the organic solvent in the mixed system is completely evaporated, the mixed system is subjected to ice bath ultrasonic treatment, and then subjected to membrane extrusion and dialysis purification to obtain a nano-preparation containing colchicine.

5. The preparation method according to claim 4, characterized in that: In (1), the ratio of the phospholipid, cholesterol, aqueous medium, surfactant and colchicine aqueous solution is (10-30) mg: (3-5) mg: (0.1-2) mL: (0.005-5) mg: (0.1-2) mL; and / or the mixed incubation conditions are: incubation at 25-65° C. for 10-30 min.

6. The preparation method according to claim 4, characterized in that: In (2), the ratio of the phospholipid, cholesterol, surfactant and colchicine aqueous solution is (10-30) mg: (3-5) mg: (0.005-5) mg: (0.1-2) mL; and / or the conditions of the ice bath ultrasonic treatment are: 100-300W ultrasonic treatment for 5 min under ice bath conditions.

7. The preparation method according to claim 4, characterized in that: The phospholipids include at least one of soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine; and / or the surfactant includes at least one of Tween 80, poloxamer and Span 20; and / or the aqueous medium includes at least one of distilled water, sodium citrate aqueous solution and ammonium sulfate aqueous solution.

8. The preparation method according to claim 3, characterized in that: The conditions for the mixing reaction of the nano preparation containing colchicine and chitosan are as follows: after mixing the nano preparation containing colchicine and chitosan solution, magnetic stirring is performed at a speed of 200 rpm for 1-24 hours; Calculated based on the colchicine content in the colchicine-encapsulated nanoformulation and the chitosan content in the chitosan solution, the mass ratio of the colchicine to the chitosan is (0.1-5) mg: (0.005-1) mg.

9. The preparation method according to claim 3, characterized in that: The conditions for the mixed reaction of the colchicine-chitosan nanoformulation and interferon-γ are as follows: after the colchicine-chitosan nanoformulation is mixed with the interferon-γ aqueous solution, it is incubated at 4° C. and 100 rpm for 4-12 hours; Calculated based on the colchicine content in the colchicine-chitosan-encapsulated nanoformulation and the interferon-γ content in the interferon-γ aqueous solution, the mass ratio of colchicine to interferon-γ is (0.1-5) mg: (0.005-1) mg.

10. Use of the colchicine preparation according to claim 1 in any of the following: (1) Application in the preparation of drugs for the treatment of liver fibrosis; (2) Application in the preparation of drugs for reversing liver fibrosis.

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