Ligand-bound copper clusters, combinations containing ligand-bound copper clusters, and their use in treating liver cirrhosis

Abstract

The present invention provides ligand-bound copper clusters (CuCs), compositions containing ligand-bound copper clusters, and uses thereof in treating patients with liver cirrhosis.

Description

[0001] This invention patent application is a divisional application based on the invention patent with application number "2020113065781", application date "November 19, 2020", and name "Ligand-bound copper clusters, combinations containing ligand-bound copper clusters and their application in the treatment of liver cirrhosis". Technical Field

[0002] The present invention generally relates to the technical field of liver cirrhosis treatment, and more particularly to ligand-bound copper clusters (CuCs), compositions containing ligand-bound copper clusters, and applications thereof in treating liver cirrhosis. Background Art

[0003] The liver is the largest solid organ in the human body and has many important functions, including: making blood proteins that help with blood clotting, transport oxygen, and aid the immune system; storing excess nutrients and returning some of them to the blood; making bile to aid in the digestion of food; helping the body store sugar (glucose) in the form of glycogen; removing harmful substances from the body, including drugs and alcohol; and breaking down saturated fats and producing cholesterol.

[0004] Cirrhosis is a slowly progressive disease that develops over many years due to long-term, ongoing liver damage. As cirrhosis progresses, healthy liver tissue is gradually destroyed and replaced by scar tissue. This scar tissue blocks blood flow through the liver and slows the liver's ability to process nutrients, hormones, medications, and natural toxins. It also reduces the liver's production of proteins and other substances. Cirrhosis may eventually lead to liver failure and / or liver cancer, which may require a liver transplant.

[0005] In the early stages of cirrhosis, the liver's compensatory function is strong and there are no obvious symptoms. Late-stage symptoms include liver damage, portal hypertension, upper gastrointestinal bleeding, hepatic encephalopathy, secondary infection, hypersplenism, ascites, cancer, and other complications. The liver gradually deforms and hardens, progressing to cirrhosis. Histopathologically, cirrhosis manifests as extensive hepatocellular necrosis, nodular regeneration of residual hepatocytes, connective tissue proliferation, and the formation of fibrous septa, leading to the destruction of the hepatic lobule architecture and the formation of pseudolobules.

[0006] Cirrhosis has different causes. Some people with cirrhosis have liver damage from multiple causes. Common causes of cirrhosis include chronic alcohol abuse, chronic hepatitis B and C infection, fatty liver disease, toxic metals, genetic disorders, nutritional imbalances, industrial toxins, medications, circulatory and metabolic disorders, cholestasis, schistosomiasis, and many others.

[0007] Cirrhosis can be diagnosed using a number of tests / techniques. For example, blood tests may indicate cirrhosis if levels of liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and bilirubin are elevated, while levels of blood proteins are low.

[0008] Currently, there is no specific cure for cirrhosis, although treatment can slow its progression by eliminating the cause. Summary of the Invention

[0009] The present invention provides for treating a subject with liver cirrhosis using ligand-bound copper clusters (CuCs), wherein the ligand-bound copper clusters comprise a copper core and a ligand, wherein the ligand binds to the copper core to form a ligand-bound copper cluster.

[0010] In some embodiments of this therapeutic use, the diameter of the copper core is 0.5-5 nm. In some embodiments, the diameter of the copper core is 0.5-3 nm.

[0011] In some embodiments of this therapeutic application, the ligand is one selected from thymine, thymine-modified hyaluronic acid (TMHA), L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0012] In some embodiments of this therapeutic use, L-cysteine and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and D-cysteine and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

[0013] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides, or cysteine-containing tetrapeptides.

[0014] In some embodiments of this therapeutic use, the cysteine-containing dipeptide is selected from L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), L(D)-histidine-L(D)-cysteine dipeptide (HC) and L(D)-cysteine-L(D)-histidine dipeptide (CH).

[0015] In some embodiments of this therapeutic use, the cysteine-containing tripeptide is selected from glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-lysine-L(D)-cysteine-L(D)-proline tripeptide (KCP) and L(D)-glutathione (GSH).

[0016] In some embodiments of this therapeutic use, the cysteine-containing tetrapeptide is selected from glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR) and glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR).

[0017] In some embodiments of this therapeutic use, the other thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine and dodecyl mercaptan.

[0018] The present invention also uses ligand-bound copper clusters (CuC) to prepare a medicament for treating liver cirrhosis in a subject. The ligand-bound copper clusters include a copper core and a ligand that binds to the copper core to form a ligand-bound copper cluster.

[0019] In some embodiments of the drug preparation and use, the diameter of the copper core is 0.5-5 nm. In some embodiments, the diameter of the copper core is 0.5-3 nm.

[0020] In some embodiments of the preparation and use of the drug, the ligand is one selected from thymine, thymine-modified hyaluronic acid (TMHA), L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0021] In some embodiments of the preparation and use of this drug, L-cysteine and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine (L-NIBC) and N-acetyl-L-cysteine (L-NAC), and D-cysteine and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine (D-NIBC) and N-acetyl-D-cysteine (D-NAC).

[0022] In some embodiments of the preparation and use of the drug, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides or cysteine-containing tetrapeptides.

[0023] In some embodiments of the preparation and use of the drug, the cysteine-containing dipeptide is selected from L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), L(D)-histidine-L(D)-cysteine dipeptide (HC) and L(D)-cysteine-L(D)-histidine dipeptide (CH).

[0024] In some embodiments of the preparation and use of the drug, the cysteine-containing tripeptide is selected from glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-lysine-L(D)-cysteine-L(D)-proline tripeptide (KCP) and L-glutathione (GSH).

[0025] In some embodiments of the preparation and use of the drug, the cysteine-containing tetrapeptide is selected from glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR) and glycine-L(D)-cysteine-(D)L-serine-L(D)-arginine tetrapeptide (GCSR).

[0026] In some embodiments of the preparation and use of the drug, the other thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine and dodecyl mercaptan.

[0027] The objects and advantages of the present invention will become apparent from the detailed description of the preferred embodiments with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Preferred embodiments of the present invention will now be described with reference to the drawings, wherein like reference numerals refer to the same elements.

[0029] Figure 1 Characterization data of L-glutathione-conjugated copper clusters (GSH-CuCs) are shown. (A) Typical transmission electron microscopy (TEM) image of GSH-CuCs. (B) Size distribution of GSH-CuCs calculated from the TEM image. (C) X-ray photoelectron spectroscopy (XPS) of 2p3 / 2 and 2p1 / 2 electrons of copper (0) in GSH-CuCs. (D) Comparison of Fourier transform infrared (FT-IR) spectra of GSH-CuCs (top) and GSH (bottom). (E) Fluorescence excitation (left) and emission spectra (right) of GSH-CuCs.

[0030] Figure 2Shown are the effects of different doses of Cu-1 and Cu-2 on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in mice with cirrhosis model. The positive control group was treated with sorafenib.

[0031] Figure 3 The results of HE staining pathological examination are shown: (A) blank control group; (B) model control group; (C) positive control group; (D) low-dose Cu-1 copper cluster administration group; (E) high-dose Cu-1 copper cluster administration group. DETAILED DESCRIPTION

[0032] The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.

[0033] Where publications are cited in this application in order to more fully describe the state of the art to which this invention pertains, the disclosures of these publications in their entireties are hereby incorporated by reference into this application.

[0034] Ligand-bound copper clusters consist of a copper core consisting of two to several hundred copper atoms and a ligand. The ligand, as a molecular component, binds to the copper core, forming a stable ligand-bound copper cluster in solution. Due to the low contrast of copper atoms, accurate measurements of the copper core using transmission electron microscopy are difficult. Generally, transmission electron microscopy indicates a core size of 0.5-5 nm.

[0035] The present invention provides copper clusters (CuCs) bound by one or more ligands, wherein a copper core is bound to one or more ligands. Binding of the ligand to the copper core means that the ligand forms a stable complex with the copper core in solution through covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, or van der Waals forces, among others. In some embodiments, the diameter of the copper core is in the range of 0.5-5 nm, preferably 0.5-3 nm, and more preferably 0.5-2.5 nm.

[0036] In some embodiments, the ligand includes, but is not limited to, thymine, thymine-modified hyaluronic acid (TMHA), L-cysteine, D-cysteine and other cysteine derivatives, such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine, N-acetyl-D-cysteine, etc.; cysteine-containing oligopeptides and their derivatives, including but not limited to cysteine-containing dipeptides, tripeptides, tetrapeptides and other peptides, such as: L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), L(D)-cysteine-L(D)-histidine ( CH), glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-glutathione (GSH), glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR), glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR), etc.; and other sulfhydryl-containing compounds, such as one or more of 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, dodecanethiol, etc.

[0037] Copper clusters bound to different ligands were synthesized according to methods reported in relevant literature (Deng 2018; Jia 2013; Wang 2013).

[0038] The present invention provides pharmaceutical compositions for treating cirrhosis of the liver in a subject. In some embodiments, the pharmaceutical composition comprises a ligand-bound copper cluster as disclosed above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is a phosphate buffered saline solution or normal saline. In some embodiments, the subject is a human. In some embodiments, the subject is a pet animal, such as a dog.

[0039] The present invention provides use of the ligand-bound copper cluster disclosed above for the manufacture of a medicament for treating liver cirrhosis in a subject.

[0040] The present invention provides uses of the ligand-bound copper clusters disclosed above for treating cirrhosis of the liver in a subject, or methods of treating cirrhosis of the liver in a subject using the ligand-bound copper clusters disclosed above. In some embodiments, the treatment method comprises administering a pharmaceutically effective amount of the ligand-bound copper cluster to the subject. The pharmaceutically effective amount can be determined by conventional in vivo studies.

[0041] The following examples are provided for the sole purpose of illustrating the principles of the present invention; they are in no way intended to limit or narrow the scope of the invention.

[0042] Example 1: Synthesis of TMHA-bound copper clusters (TMHA-CuCs)

[0043] 10mL of TMHA (DS 10.5%) solution (0.1mM, pH 7.0) was gradually heated to 37°C to dissolve the TMHA. 2mL of copper sulfate (20mM, pH 7.0) solution was added dropwise and the reaction was continued at 37°C in the dark for another 20 minutes. Under ultraviolet light (365nm), bright orange-red emission was clearly visible, indicating the successful synthesis of TMHA-bound copper clusters. Finally, the resulting solution was stored at 4°C in the dark for later use. The copper core diameter of the spherical TMHA-CuCs was 1.64±0.48nm.

[0044] Example 2: Synthesis and Identification of Ligand-Bound Copper Clusters with Different Ligands

[0045] 2.1 Synthesis of L-glutathione (GSH)-bound copper clusters

[0046] Add 500mg of glutathione (GSH) to 50mL of water and, while slowly stirring, add 20mL of a 5mM Cu(NO3)2 solution. The solution quickly produces a white suspension. Slowly heat the mixture to 50-60°C for 20 minutes. Add 1M NaOH solution dropwise until the solution turns pale yellow and clear. Cool the product to room temperature and precipitate it by adding several times the volume of ethanol. Repeat this process three times.

[0047] 2.2 Synthesis of L-cysteine-bound copper clusters

[0048] Under vigorous stirring, slowly add 50 mL of 10 mM CuCl2 dropwise to a freshly prepared L-cysteine solution (50 mL, 10 mM). After approximately 30 minutes, slowly add 0.5 mL of 1 M NaOH dropwise to the solution. Continue the reaction for 2 hours. Centrifuge the product at 8000 rpm for 20 minutes, and store the supernatant at 4°C in the dark.

[0049] 2.3 Synthesis of PEG-conjugated copper clusters

[0050] 2.5 g of PEG-SH (molecular weight 2000 or 5000) was dissolved in 100 ml of ultrapure water at room temperature. With vigorous stirring, 4 mL of a 0.5 M Cu(NO) solution was added dropwise. The mixture was stirred at room temperature until the color faded and gradually turned milky white. The hydrogel was gradually heated to 80°C for 15 minutes. A 3 M NaOH solution was added dropwise until the solution became clear and transparent. The product was centrifuged at 8000 rpm for 20 minutes and lyophilized in a freeze dryer to obtain a solid sample.

[0051] 2.4 Synthesis of Ligand-Bound Copper Clusters with Other Ligands

[0052] Ligand-bound copper clusters with other ligands can also be synthesized using the above method; the specific synthesis method needs to be slightly modified in some solvents and operations; other ligands include thymine, L(D)-cysteine and other cysteine derivatives, such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine and N-acetyl-D-cysteine, cysteine-containing oligopeptides and their derivatives, including but not limited to dipeptides, tripeptides, tetrapeptides and other cysteine-containing peptides, such as L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), L(D)-cysteine-L(D) -histidine (CH), glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-glutathione (GSH), glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR), glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR) and other thiol-containing compounds, such as one or more of 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiol, D-3-trolovirol and dodecyl mercaptan.

[0053] 2.5 Identification of ligand-bound copper clusters

[0054] As an example, the following is the characterization data of L-GSH-bound copper clusters (GSH-CuCs).

[0055] 1) Morphological observation by transmission electron microscopy (TEM)

[0056] The test powder (GSH-CuCs sample) was dissolved in ultrapure water to a concentration of 2 mg / L. The sample was then prepared using the hanging drop method. Specifically, 5 μl of the sample was dropped onto a copper grid and allowed to evaporate naturally until the droplet disappeared. The sample morphology was then observed using a JEM-2100F STEM / EDS field emission high-resolution transmission electron microscope.

[0057] Figure 1 Panels A and B show typical SEM images of GSH-CuCs, and their size distribution was calculated from different TEM images. The results show that GSH-CuCs are well dispersed, with sizes ranging from 0.5 to 5.0 nm.

[0058] 2) X-ray photoelectron spectroscopy

[0059] X-ray photoelectron spectroscopy (XPS) was measured on an ESCALAB 250xi X-ray photoelectron spectrometer. Double-sided conductive tape (3 mm × 3 mm) was attached to aluminum foil. The test powder was evenly applied to the double-sided tape and covered with a layer of aluminum foil. The sample was held at 8 MPa for 1 minute. Any residual powder on the surface was removed, and then a central sample (1 mm × 1 mm) was cut out for XPS analysis.

[0060] Figure 1 The C amplitude is the XPS spectrum of copper in GSH-CuCs. The two peaks appear at 931.98 and 951.88 eV, which can be attributed to the 2p 3 / 2 and 2p 1 / 2 The binding energy of electrons. There is no Cu 2p near 942.0 eV 3 / 2 The satellite peaks confirm the absence of Cu(II) electrons. Since the binding energy of Cu(0) is only 0.1 eV away from that of Cu(I), it is impossible to exclude the formation of Cu(I), and the valence state of copper in the obtained GSH-CuCs is likely to be between 0 and +1.

[0061] 3) Fourier transform infrared spectroscopy

[0062] FT-IR spectra were measured on a PerkinElmer LS 55 fluorescence spectrometer. The test powder was dissolved in ultrapure water and measured at room temperature. The scanning range was 200-800 nm, the sample cell was a standard quartz cuvette, and the optical path length was 1 cm.

[0063] Figure 1 Panel D shows the comparison of FT-IR spectra of GSH-CuCs (upper) and GSH (lower). GSH exhibits many characteristic infrared bands, namely COOH (1390 and 1500 cm -1 ), NH stretching (3410cm -1) and NH2 group NH bending (1610cm -1 ). At 2503cm -1 The peaks observed at can be attributed to the SH stretching vibration mode. -1 ), GSH-CuCs also exhibited corresponding infrared characteristics. The results showed that the SH bond was broken and the GSH molecule was bound to the surface of the copper core through the formation of copper-sulfur bonds.

[0064] 4) Fluorescence spectroscopy

[0065] The test powder was dissolved in ultrapure water and measured by fluorescence spectrometry at room temperature.

[0066] like Figure 1 As shown in the E-amplitude, under excitation peak at 365 nm, the copper clusters exhibit red emission with a peak at 595 nm and a corresponding full width at half maximum (FWHM) of approximately 80 nm. Notably, the FL intensity of GSH-CuCs significantly increases when ethanol is added to the solution due to aggregation-induced emission enhancement. Furthermore, the large Stokes shift (230 nm) demonstrates promising applications as fluorescent probes and bioimaging.

[0067] Example 3: Animal Experiment

[0068] 3.1 Experimental Materials and Animals

[0069] 3.1.1 Test samples

[0070] Cu-1: GSH-modified copper clusters (GSH-CuCs), with a size of 0.5-5 nm.

[0071] Cu-2: Cysteine (Cys)-modified copper clusters (Cys-CuCs), with a size of 0.5-5 nm.

[0072] All test samples were synthesized using the above method with slight modifications, and their quality was identified using the above method.

[0073] 3.1.2 Positive control samples

[0074] Sorafenib.

[0075] 3.1.3 Experimental animals and groups

[0076] Seventy SPF male C57BL / 6N mice (purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd. (production license number: SCXK (Beijing) 2019-0008), 6-8 weeks old, weighing 16-20 g, were randomly divided into 7 groups (n=10) according to body weight: blank control group, model group, positive control group, Cu-1 low-dose group, Cu-1 high-dose group, Cu-2 low-dose group, and Cu-2 high-dose group.

[0077] 3.2 Modeling Method

[0078] Except for the blank control group, all other groups of mice were treated with carbon tetrachloride (CCl4)-induced liver cirrhosis models. The modeling method was as follows: (1) Each mouse was intraperitoneally injected with 10% CCl4 (diluted with olive oil) at 7 μL / g body weight twice a week for 8 weeks; the control group mice were intraperitoneally injected with an equal amount of olive oil solvent. (2) Starting from the 6th week, 2 mice were selected and sacrificed 48 hours after the last injection each week. The appearance of the mouse liver was observed. When the appearance was consistent with the characteristics of liver cirrhosis (8th week), the liver tissue was formalin-fixed and HE staining and Masson staining were performed to evaluate the establishment of the liver cirrhosis model.

[0079] 3.3 Administration

[0080] After successful model establishment, mice in the positive control group were gavaged with 25 mg / kg sorafenib. The low- and high-dose Cu-1 and Cu-2 groups were intraperitoneally injected with the corresponding test products at doses of 2.5 or 10 mg / kg, respectively. Mice in the blank control and model groups were intraperitoneally injected with normal saline at 10 mL / kg. Dosing was performed once daily for 20 consecutive days.

[0081] 3.4 Biochemical testing

[0082] After dosing, mice were bled from their orbits and serum collected. Five indicators, including albumin (ALB), total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and monoamine oxidase (MAO), were assayed using a Zhongsheng Beikong kit and a biochemical analyzer (Siemens). The assays were performed strictly according to the kit instructions.

[0083] Table 1 shows the product information of the kits used for biochemical detection

[0084]

[0085] 3.5 Pathological examination

[0086] 3.5.1 HE staining

[0087] After euthanasia, liver tissue samples were fixed in 4% paraformaldehyde for at least 48 hours. Following fixation, they were dehydrated using a gradient of alcohol and cleared using xylene and ethanol. The liver tissue was then waxed and embedded. After the embedded material was trimmed, glued, and reshaped, the liver tissue was sectioned using a paraffin microtome at a thickness of 4 μm. The main HE staining procedure was as follows: after baking the sections in a 65°C oven, the sections were treated with xylene and then dehydrated using a gradient of ethanol. After staining with hematoxylin, blueing solution, and 0.5% eosin solution, the sections were then treated with a gradient of ethanol and xylene and mounted with neutral gum. Liver fibrosis was observed microscopically.

[0088] 3.5.2 Masson staining

[0089] Mouse liver tissue sections were baked, dewaxed, and dehydrated. After chromatinization, sections were stained with Regaud's hematoxylin. After washing, sections were stained with Masson's Ponceau-Fuchsin solution. Sections were rinsed with 2% glacial acetic acid and differentiated with 1% phosphomolybdic acid. Sections were stained directly with aniline blue or light green solution and briefly rinsed with 0.2% glacial acetic acid. Sections were cleared with 95% alcohol, anhydrous alcohol, and xylene, and then mounted with neutral gum. Liver tissue was observed under a microscope.

[0090] 3.6 Experimental Results

[0091] 3.6.1 Successful modeling

[0092] The livers of the model mice were divided into circular or oval masses of varying sizes by proliferating fibrous septa. Serum ALT, TBil, and AST levels were significantly elevated compared to the blank control group, while ALB levels were significantly decreased. MAO levels were not significantly different from the blank control group, but were also elevated. These results indicate that the model was successfully established.

[0093] 3.6.2 Effects of the trial drug on alanine aminotransferase (ALT), total bilirubin (TBil), aspartate aminotransferase (AST), monoamine oxidase (MAO), and albumin (ALB)

[0094] Depend on Figure 2As shown in Figure 1, ALT activity in the model group was significantly elevated compared to that in the blank control group (from an average of 43.5±8.1 U / L to 188.5±4.9 U / L, P<0.01), suggesting that liver function in cirrhosis mice was impaired. Compared to the model group, low and high doses of Cu-1 and Cu-2 (minimum 37.0±5.7 U / L, maximum 38.6±5.6 U / L) as well as the positive control (42.8±5.4 U / L) significantly reduced ALT activity to the level of the blank control group (P<0.01).

[0095] Depend on Figure 2 As shown in Figure 2, the AST activity in the model group was significantly increased compared with that in the blank control group (from 141.8±13.5U / L to 192.0±11.3U / L, P<0.05). High-dose administration of Cu-1 and Cu-2 significantly reduced AST activity to 146.3±8.4U / L or 144.3±8.1U / L, respectively, which was the same level as the blank control group (141.8±13.5U / L) and significantly different from the model control group (both P<0.01). The positive control group also reduced AST activity ((165.5±11.6U / L, P<0.05), but the reduction was less than that in the high-dose Cu-1 and Cu-2 groups.

[0096] Depend on Figure 2 As shown in Figure 3, the TBil concentration in the model group was significantly elevated compared to that in the blank control group (from 1.02±0.20μmol / L to 2.91±0.39μmol / L), with a significant difference from the blank control group (P<0.01). Compared with the model group, both low- and high-dose administration of Cu-1 and Cu-2 significantly reduced serum TBil (maximum 1.16±0.30μmol / L, minimum 1.08±0.08μmol / L, both P<0.01), approaching the level of the blank control group (1.02±0.20μmol / L).

[0097] Depend on Figure 2 D shows that MAO activity in the model group was slightly higher than that in the blank control group (blank control group: 18.8±2.9U / L, model group: 21.5±0.7U / L), but the difference was not statistically significant, suggesting that changes in MAO activity in mice with carbon tetrachloride-induced cirrhosis were not significant. However, compared with the model group, high doses of Cu-1 and Cu-2 significantly reduced serum MAO levels to 17.3±1.5U / L (P<0.01) or 18.3±2.1U / L (P<0.05), respectively, demonstrating superior effects to the positive control.

[0098] Depend on Figure 2E shows that the ALB level in the model group showed a significant decrease compared to the ALB level in the blank control group (from 24.2±0.6g / L to 22.1±1.3g / L), with a significant difference from the blank control group (P<0.05), indicating that carbon tetrachloride treatment can significantly reduce serum ALB levels. However, Cu-1 and Cu-2 did not significantly affect serum ALB levels.

[0099] The above results show that the ligand-bound copper clusters reduce the levels of ALT, AST, TBIL and MAO in a dose-dependent manner, suggesting that the liver function of mice is restored. Its effect is better than that of the positive control drug at least in some indicators.

[0100] 3.6.3 Pathological examination

[0101] Liver cirrhosis is characterized by diffuse fibrosis and pseudolobule formation in liver tissue. HE staining results show that Figure 3 As shown in A, the normal liver tissue structure of mice in the blank control group is clear, the liver lobules are intact, the liver cell cords are neatly arranged and radially arranged with the central vein as the center, the liver cell nuclei are normal, and there is only a small amount of fibrous tissue in the portal area; Figure 3 As shown in B, in the liver of the model control group mice, the hepatocytes were arranged in disorder, ballooning occurred, the hepatic lobules almost disappeared, and the pseudolobules ( Figure 3 The right arrow in B) appears in large numbers, and collagen fibers proliferate and form round or oval fiber septa ( Figure 3 Leftward arrow in B). Compared with the model control group, Figure 3 As shown in C, in the positive control group, the degree of liver damage was significantly reduced, the cells were arranged in a regular pattern, and although collagen fibers were proliferating, they were significantly weakened, no fibrous septa were formed, and pseudolobules almost disappeared. However, compared with normal liver tissue, the gaps between liver cells in the positive control group were significantly enlarged ( Figure 3 (Downward arrow in C). Compared with the model control group, the hepatocytes in the two copper cluster drug (Cu-1 and Cu-2) drug groups showed extremely significant recovery from liver injury, as evidenced by a significant reduction in fibrosis and pseudolobules in the liver, which was dose-dependent.

[0102] Figure 3 D and Figure 3 E shows the HE staining images of the effects of low-dose and high-dose administration on liver tissue damage and repair, represented by Cu-1 copper clusters. Figure 3 As shown in D, in the low-dose Cu-1 copper cluster group, the hepatocytes were arranged more neatly, the pseudolobules almost disappeared, and the collagen fiber proliferation was significantly weakened. However, the intercellular spaces between the liver cells were enlarged to a certain extent compared with normal liver tissue ( Figure 3 D). Figure 3As shown in Figure E, the high-dose Cu-1 copper cluster group showed a more pronounced improvement than the low-dose group, with complete disappearance of pseudolobules, minimal collagen fiber proliferation, and almost no visible enlargement of the intercellular spaces between liver cells, showing no significant difference from normal liver cells. This demonstrates that the Cu-1 copper cluster drug demonstrates a superior effect on repairing liver tissue damage compared to the positive control drug.

[0103] The results of Masson staining were consistent with those of HE staining.

[0104] Cu-2 copper cluster drugs also showed similar effects to Cu-1 copper cluster drugs.

[0105] In summary, the two copper cluster test drugs, Cu-1 and Cu-2, significantly reduced liver fibrosis and hepatic pseudolobules. Liver function test results also showed signs of liver function recovery. Changes in alanine aminotransferase (ALT) and total bilirubin (TBil) were the most significant. Aspartate aminotransferase (AST) and monoamine oxidase (MAO) also showed significant recovery, while albumin (ALB) changes were less pronounced. The two copper cluster test drugs significantly improved liver function and liver pathology in cirrhotic mice, and their overall effect was superior to that of the positive control, sorafenib, providing experimental evidence for further application.

[0106] The same procedure was also used to experiment with GSH-CuCs and Cys-CuCs of different sizes and CuCs bound to other ligands, and the results were similar, so they will not be described in detail here.

[0107] Although the present invention has been described with reference to specific embodiments, it will be understood that the embodiments are illustrative and that the scope of the invention is not limited thereto. Alternative embodiments of the present invention will become apparent to those skilled in the art to which the present invention relates. Such alternative embodiments are considered to be within the spirit and scope of the present invention. Accordingly, the scope of the present invention is described by the appended claims and supported by the foregoing description.

[0108] References

[0109] Deng HHet al.An ammonia-based etchant for attaining coppernanoclusters with green fluorescence emission.Nanoscale,2018,10,6467.

[0110] Jia X.et al.Cu Nanoclusters with Aggregation Induced EmissionEnhancement.Small,2013,DOI:10.1002 / smll.201300896.

[0111] Wang C.and Huang Y.GREEN ROUTE TO PREPARE BIOCOMPATIBLE AND NEARINFRARED THIOLATE-PROTECTED COPPER NANOCLUSTERS FOR CELLULAR IMAGING.NANO:Brief Reports and Reviews.2013,8(5):1350054(10 pages).

Claims

1. Use of ligand-bound copper clusters in the preparation of a medicament for treating patients with liver cirrhosis, characterized in that: The ligand-bound copper clusters include: Copper core; and a ligand, wherein the ligand binds to the copper core to form a ligand-bound copper cluster; The ligand is selected from L-glutathione or D-glutathione.

2. The use according to claim 1, characterized in that The diameter of the copper core is 0.5-5 nm.

3. The use according to claim 1, characterized in that The diameter of the copper core is 0.5-3 nm.

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