Use of vidarabine phosphate in the preparation of a medicament for treating liver ischemia-reperfusion injury

Abstract

The application discloses application of vidarabine phosphate in preparation of a medicine for treating liver ischemia-reperfusion injury, and relates to the field of biological medicines. The application finds that vidarabine phosphate can obviously reduce serum liver enzyme levels, obviously reduce GZMB accumulation in liver tissues and expression of inflammation-related factors including Il-1beta, Tnf-alpha and Mmp9, indicates that liver inflammation reaction is reduced, and reduces liver cell apoptosis-related protein cleaved-caspase 3 levels and liver histological injury. In summary, the application first finds application of vidarabine phosphate in treating liver ischemia-reperfusion injury, which can play a role in improving liver enzyme levels and reducing liver inflammatory injury, and has wide clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of vidarabine phosphate in preparation of a medicine for treating liver ischemia-reperfusion injury. BACKGROUND

[0002] Liver ischemia-reperfusion injury (IRI) occurs when there is a sharp decrease or interruption in blood supply to the liver, and the low-oxygen environment of the liver tissue caused by insufficient blood supply to the organ. In the case of ischemia and hypoxia, liver function is significantly inhibited and damaged, and subsequent reoxygenation of the tissue by blood flow reperfusion will trigger more severe oxidative stress and inflammatory response, further causing liver damage. IRI is an inevitable process during liver transplantation or liver resection surgery, and is an important cause of postoperative graft dysfunction and liver failure.

[0003] Liver IRI is a complex process, including damage caused by hypoxia and sterile inflammation mediated by immune imbalance. Innate immune cells, including Kupffer cells and neutrophils, have been reported as damage-associated molecular pattern-responsive cells. NK cells account for 50% of the lymphocyte population and are the most numerous lymphocyte population in the human liver, and mainly participate in innate immune responses. NK cells play a key role in antiviral and antitumor responses, but their role in liver IRI is not yet clear. Arias-Diaz et al. proved that NK cell infiltration significantly increased in a rat model of liver IRI (Arias-Diaz J, Ildefonso JA, JJ. et al. Both tacrolimus and sirolimus decrease Th 1 / Th2 ratio. and increase regulatory T lymphocytes in the liver after ischemia / reperfusion. Laboratory investigation; a journal of technical methods and pathology, 2009, 89(4): 433-445.) Previous studies have shown that depletion of NK cells can significantly reduce the secretion levels of liver inflammatory factors (TNF-a, IL-1b, IL-6, IL-8 and CXCL-2), and reduce neutrophil infiltration in the early stage of liver IRI. NK cells can exert cytotoxic effects after activation by releasing perforin, granzyme B (GZMB) and pro-inflammatory compounds such as IFN-γ and TNF-α. GZMB is the most abundant type of cells, which can cause apoptosis through multiple pathways such as cutting and activating several pro-apoptotic proteins after infection in the pathway of perforin-dependent cytotoxicity. It has been previously identified as a major toxic protein in ischemic diseases such as stroke and myocardial infarction. Santos-Zas et al. found that GZMB deficiency can reduce apoptosis and pro-inflammatory cytokine expression, limiting cardiac damage after ischemia (Santos-Zas I, Lemarié J, Zlatanova I, et al. Cytotoxic CD8(+) T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling. Nat Commun, 2021, 12(1): 1483.). Our research results show that NK cells with high GZMB expression play a role in graft injury in mouse liver IRI models and hepatocyte hypoxia-reoxygenation models. In addition, genetic intervention or pharmacological inhibition of NK cell-derived GZMB leads to reduced liver IRI in vivo.

[0004] Vidarabine phosphate (Vida) is a monophosphorylated compound of vidarabine, which has high solubility and also has anti-viral and possibly anti-tumor properties. Vidarabine phosphate has the therapeutic advantage of intermittent intramuscular or intravenous injection. However, there is no report on the prevention and treatment of liver ischemia-reperfusion injury by vidarabine phosphate. SUMMARY

[0005] In order to solve the above technical problems, the application aims to provide a new application of vidarabine phosphate in treating liver ischemia-reperfusion injury.

[0006] The specific technical scheme of the application is:

[0007] In one aspect, the application provides an application of vidarabine phosphate in preparing a medicament for treating liver ischemia-reperfusion injury.

[0008] Specifically, the liver ischemia-reperfusion injury is liver injury caused by post-ischemia reperfusion after liver transplantation or liver resection.

[0009] The application provides an application of vidarabine phosphate in preparing a medicament for treating liver ischemia-reperfusion injury.

[0010] Preferably, the medicament is an injection preparation, and the administration dose is 100 mg / kg.

[0011] In another aspect, the application further provides a medicament for treating liver ischemia-reperfusion injury, which is vidarabine phosphate itself and a pharmaceutically acceptable salt.

[0012] Preferably, the medicament is an injection preparation, and the administration dose is 100 mg / kg.

[0013] In the application process, the inventors constructed a mouse liver ischemia-reperfusion model and used vidarabine phosphate to treat liver ischemia-reperfusion injury of the mouse.

[0014] In the research of vidarabine phosphate in improving liver ischemia-reperfusion injury, the usage and dosage are as follows: 12 hours before ischemia, the mouse of 8 weeks old is injected with the medicament through the tail vein once, and the dosage is 100 mg / kg of body weight.

[0015] The application has the following beneficial effects:

[0016] The application finds that vidarabine phosphate can obviously reduce the serum liver enzyme level, obviously reduce the accumulation of GZMB in liver tissue and the expression of inflammation-related factors including I1-1β, Tnf-α and Mmp9, indicating that the liver inflammation reaction is reduced, and the level of apoptosis-related protein cleaved-caspase 3 in the liver is reduced, and the liver histological injury is reduced. The research results of the application provide a theoretical basis for vidarabine phosphate in improving liver ischemia-reperfusion injury, especially the finding that the drug vidarabine phosphate, which has been used in clinical treatment but is limited to treating viral infection-related diseases, can be applied to liver ischemia-reperfusion injury, which is an old drug with a new use and provides a new strategy for treating liver ischemia-reperfusion injury.

[0017] In summary, the application first discovers the application of vidarabine phosphate in treating liver ischemia-reperfusion injury, which can play a role in improving liver enzyme levels and reducing liver inflammatory injury, and has wide clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Computer molecular dynamics simulation experiment of FDA-approved vidarabine phosphate and human or mouse GZMB. A: Three-dimensional structure of mouse GZMB protein and small molecule vidarabine phosphate, wherein the active site Ser203 in the catalytic pocket of mouse GZMB and vidarabine phosphate is represented by a stick; B: Distance between the phosphate group of vidarabine phosphate and the hydroxyl group of Ser203 over time. Inset: MD simulation of vidarabine phosphate and mouse GZMB; C: Mouse GZMB contact potential; D: Time evolution of the interaction energy of vidarabine phosphate and mouse GZMB.

[0019] Figure 2 Vidarabine phosphate reduces GZMB accumulation after ischemia-reperfusion, improves liver enzyme indicators, and reduces liver histological damage. A: Overall model construction idea; B: Serum GZMB level in mice; C: Intrahepatic GZMB level determined by ELISA method; D: Serum liver enzyme level in mice.

[0020] Figure 3 Immunohistochemical staining of GZMB and Cleaved-caspase3 (C-cas3) (scale bar = 100 pm) and HE staining of liver tissue (scale bar = 100 pm, dotted line depicts necrotic area). *p<0.05, **p<0.01.

[0021] Figure 4 Vidarabine phosphate reduces liver inflammatory response and apoptosis after ischemia-reperfusion. A: Relative expression amount of inflammatory factors Il-1β, Tnf-α and Mmp9 in mouse liver; B: Western blot detection of Bax and Bcl2 protein levels in liver. *p<0.05. DETAILED DESCRIPTION

[0022] Example 1: Theoretical modeling and molecular dynamics (MD) simulation

[0023] The 3D structure of GZMB protein was generated using AlphaFold 2. Subsequently, molecular dynamics (MD) simulation was performed on this model for the purpose of equilibration. Small molecule ligands arranged within the catalytic site of the enzyme were calculated by AutoDock Vina for molecular docking and dynamics simulation to predict the interaction of mouse GZMB with GZMB inhibitor candidate drugs (i.e. tannic acid, mupirocin, cefoperazone, biphenyl amide benzoic acid and vidarabine phosphate). Further we performed MD simulation to check the stability and behavior of the resulting complex. The MD simulation framework was assembled and executed using GROMACS, and the trajectories generated were rigorously analyzed by visual molecular dynamics (VMD) software.

[0024] From Figure 1 As can be seen from A, GZMB, as a serine protease, has a catalytic pocket that binds substrates, and there is a conserved serine residue in the pocket. During all MD simulations, only vidarabine phosphate remained bound to mouse GZMB, indicating a strong interaction between them. Figure 1 The curve in B shows the distance between the phosphate group of vidarabine phosphate and the hydroxyl group of serine 203 in the simulation trajectory. Figure 1 The electrostatic potential surface of the protein is plotted in C. The results show that the vidarabine phosphate group plays a key role in the binding of vidarabine phosphate and GZMB, Figure 1 D shows the interaction energy of vidarabine phosphate with mouse GZMB, confirming that there is an electrostatic interaction between vidarabine phosphate and GZMB.

[0025] Example 2: Establishment of mouse liver ischemia-reperfusion model

[0026] I. Experimental animals

[0027] Experimental animals were grouped and the model was established: 8-week-old SPF male C57BL / 6 mice with an average weight of 20-30 g in good condition were all raised in SPF animal rooms. This study was approved by the Zhejiang Province Experimental Animal Center Experimental Animal Welfare Ethics Committee, with ethical moral approval number ZJCLA-IACUC-20040168.

[0028] II. Grouping of experimental mice

[0029] Before establishing the model mice, according to the principle of random allocation, the mice were randomly divided into sham operation group: Sham group; Ischemia-reperfusion experimental group: IRI+PBS group (NC group); IRI+cytarabine (Cytarabine, Cyta group); IRI+vidarabine phosphate treatment group (Vida group).

[0030] III. Construction of mouse liver ischemia-reperfusion model

[0031] Mice were anesthetized with 1 wt% pentobarbital (60 mg / kg) intraperitoneally, fixed on a clean surgical table, and the abdominal cavity was cut open with surgical scissors. The skin of the abdominal cavity was fixed with hemostatic forceps to fully expose the hepatic portal area. The portal vein and hepatic artery supplying the liver were separated, and a non-invasive vascular clamp was used to block the blood flow of the left and middle liver lobes. The abdominal cavity was temporarily closed with a warm gauze, and the mouse was placed on a constant temperature pad. After 90 minutes, the vascular clamp was removed, and the mouse’s liver blood supply was restored. The abdominal cavity was sutured and closed. Sham operation group mice underwent the same operation except for the vascular clamp. The animals were placed on a warm pad maintained at 37°C until fully awake. Serum and liver tissue samples were collected for subsequent experiments.

[0032] Four, drugs and treatments

[0033] Compound vidarabine phosphate was purchased from Shanghai Taoshu Biological Technology Co., Ltd. (Product No. T20048, CAS: 29984-33-6) and dissolved in PBS. As shown in Figure 2 A, vidarabine phosphate was intraperitoneally injected into mice at a dose of 100 mg / kg body weight 12 hours before the operation. PBS was injected in the same way as the negative control, and cytarabine was injected intravenously as a drug intervention control to avoid interference with the effects of antiviral drugs.

[0034] Four, sample collection and processing

[0035] 1. Sample collection time: sample collection at 6 hours after reperfusion.

[0036] 2. Collect mouse peripheral serum samples to detect liver function related indicators:

[0037] After the collected blood coagulates, centrifuge at 1000 rpm, 4°C for 15 min, separate the serum, and store at -80°C after aliquoting.

[0038] The concentration of serum GZMB was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp, #SEA600Mu). The results are shown in Figure 2 B, there was no significant difference in serum GZMB levels among the three groups after liver IRI.

[0039] The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were determined using an automatic biochemical analyzer (Chemray 800, Rayto, Shenzhen, China), provided by the Affiliated Hospital of Zhejiang University. The results are shown in Figure 2 D, compared with other IRI groups, the serum ALT and AST levels of the vidarabine phosphate group were significantly reduced, indicating that vidarabine phosphate has the effect of improving liver function after liver IRI.

[0040] 3. Collect mouse liver samples to detect histological indicators

[0041] After 90 minutes of ischemia and 6 hours of reperfusion, the mice were sacrificed by cervical dislocation. The chest cavity was opened from the abdominal white line of the mouse using ophthalmic scissors, the liver tissue was exposed, the gallbladder was removed, and the left and middle lobes of the liver were removed. A piece of tissue (1 cm x 1 cm x 1 cm) was taken 4 mm from the liver margin, placed in a 4% paraformaldehyde solution for fixation for HE staining, and the remaining liver tissue was cut into several pieces (1 cm x 1 cm x 1 cm) and stored in a -80°C freezer for later use.

[0042] 3.1 The concentration of GZMB protein in the tissue was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp, #SEA600Mu). The results are shown in Figure 2 C shows that after liver IRI, the accumulation of GZMB in the liver tissue of the phospho-ara-A treatment group was significantly reduced.

[0043] 3.2 Total RNA was extracted from the liver tissue and reverse transcribed into cDNA for RT-qPCR experiments to detect the gene expression levels of inflammatory factors Il-1β, Tnf-α, and Mmp9, as shown in Figure 4 A shows that after liver IRI, the phospho-ara-A treatment group reduced the inflammatory response in the liver by inhibiting GZMB, while there was no significant anti-inflammatory effect after ara-C treatment.

[0044] 3.3 Total protein was extracted from the liver tissue for Western blot experiments to detect the expression levels of apoptosis-related proteins BAX and BCL2. As shown in Figure 4 B shows that the phospho-ara-A treatment group increased the expression of the pro-apoptotic protein BAX, which is protective, and decreased the expression of BCL2. There was a significant difference in the BCL2 / BAX level, and there was no significant decrease in the apoptosis level after ara-C treatment.

[0045] 4. Tissue immunohistochemical staining

[0046] Liver tissue specimens were fixed with 4% paraformaldehyde solution, dried and then immersed in paraffin. Sections of 5 μm thickness were obtained, deparaffinated with xylene and then rehydrated through an ethanol gradient. The degree of liver necrosis was evaluated with hematoxylin and eosin (HE) staining. Immunohistochemistry (IHC) was used to evaluate GZMB (abcam, #255598), Cleaved-caspase3 (Service Bio, #GB11532). Slides were examined under a SLIDEVIEW digital slide scanner (VS200, Olympus, Japan). Necrotic areas in randomly selected fields on each HE slide were represented by loss of architecture, vacuolization, increased karyorrhexis and increased acidophilic granulation. Two pathologists independently assessed histological criteria of liver injury using Suzuki's score (congestion, vacuolization and necrosis). The percentage of positive areas in representative views of IHC sections was statistically analyzed using ImageJ software.

[0047] Results are shown in Figure 3 As shown in the results, the liver lobules in the sham group were complete, with clear boundaries and complete and full hepatocyte structure; while in the IRI group, liver cells were necrotic, with increased infiltration of inflammatory cells such as neutrophils and monocytes, and obvious vascular obstruction. After treatment with vidarabine phosphate, the accumulation of GZMB in liver tissue was significantly reduced, the expression of C-cas3 was reduced, and the liver tissue damage was improved. However, no significant protective effect was observed after treatment with cytarabine.

[0048] In summary, the present application proposes the use of vidarabine phosphate in the preparation of a drug for treating liver ischemia-reperfusion injury, and clarifies that vidarabine phosphate can improve liver enzyme levels after IRI by inhibiting the expression of GZMB, reduce liver inflammatory response and cell apoptosis, and this protective effect is unrelated to the effect of antiviral drugs, providing a new treatment strategy for the clinical treatment of liver ischemia-reperfusion injury.

[0049] Finally, it should be noted that the above-mentioned embodiments are only some specific embodiments of the present application, and the present application is not limited to the described embodiments. In addition, those skilled in the art can make different modifications to the present application without departing from the spirit of the present application, and all these equivalent forms are considered to fall within the scope of protection defined by the present application.

Claims

1. The use of adenosine phosphate in the preparation of a medicament for treating hepatic ischemia-reperfusion injury, wherein the hepatic ischemia-reperfusion injury is liver damage caused by ischemia-reperfusion after liver transplantation or liver resection, and the medicament is administered before hepatic ischemia-reperfusion.

2. The application as described in claim 1, characterized in that, The drug is an injectable preparation.

3. The application as described in claim 1, characterized in that, The dosage is 100 mg / kg.

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