Application of sunitinib in preparation of medicine for treating secondary injury caused by intracranial hemorrhage

Abstract

The invention discloses an application of sunitinib in preparation of a medicine for treating secondary injury caused by intracranial hemorrhage. It is found for the first time that sunitinib can act on a CSF-1R / PI3K / Akt axis, pathological activation and pro-inflammatory response of microglial cells are effectively inhibited, the normal phagocytic function and lipid metabolism balance of the microglial cells are remarkably recovered, myelin sheath damage around hematoma is effectively relieved, and the neurological function score, the movement coordination ability and the exploration behavior of ICH mice are remarkably improved; the progress of secondary injury caused by intracranial hemorrhage is hindered from multiple dimensions, the curative effect is superior to that of a positive control drug pecetinib (PLX3397), and a brand new molecular target and an interference strategy are provided for comprehensive treatment of ICH.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of sunitinib in the preparation of a therapeutic drug for secondary injuries caused by intracranial hemorrhage. Background Technology

[0002] Intracranial hemorrhage (ICH) is one of the most destructive cerebrovascular emergencies, with extremely high mortality and disability rates, posing a serious challenge to global public health. Statistics show that the incidence of ICH in China is approximately 60–80 per 100,000 people, with a compound annual growth rate of 2.9%. With the accelerating aging of the population and the prevalence of unhealthy lifestyles, the disease burden of ICH is expected to continue to increase, placing increasingly heavy pressure on healthcare systems.

[0003] Neurological damage caused by ICH mainly includes two stages: primary injury and secondary injury. Primary injury is caused by the mechanical compression and direct damage to brain tissue caused by the hematoma itself. Although the impact can be alleviated by surgical removal of the hematoma, a large amount of clinical evidence shows that surgical intervention has not significantly improved the long-term neurological prognosis of patients. Approximately 80% of ICH survivors still have severe neurological deficits six months after onset, highlighting the obvious limitations of current treatment strategies that focus on treating primary injury.

[0004] Secondary injury plays a crucial role in the pathological progression of ICH, with microglia, as the main immune cells in the central nervous system, being considered the core mechanism driving this injury. During the acute phase of ICH, microglia around the hematoma rapidly transform into a disease-associated phenotype, characterized by the massive release of pro-inflammatory factors, impaired phagocytic function, and metabolic reprogramming, thereby exacerbating neuroinflammatory responses and hindering tissue repair. Although intervention strategies targeting microglia pathological activation theoretically hold the promise of blocking the progression of secondary injury, no related treatments have yet been successful in clinical trials.

[0005] Therefore, there is an urgent need in this field to develop novel treatment strategies that can precisely regulate microglial function and inhibit its pathological activation in order to effectively reduce secondary damage after ICH and ultimately improve the neurological prognosis of patients. Summary of the Invention

[0006] The purpose of this invention is to provide the application of sunitinib in the preparation of a therapeutic drug for secondary injuries caused by intracranial hemorrhage, and to propose a new treatment option for alleviating secondary injuries after ICH.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] The use of sunitinib in the preparation of therapeutic drugs for secondary injuries caused by intracranial hemorrhage, wherein the chemical structural formula of sunitinib is:

[0009]

[0010] Compared with the prior art, the technical effects of the present invention are reflected in:

[0011] (1) This invention is the first to discover that sunitinib can act on the CSF-1R / PI3K / Akt axis, effectively inhibit the pathological activation and pro-inflammatory response of microglia, significantly restore the normal phagocytic function and lipid metabolism balance of microglia, effectively reduce myelin damage around hematoma, significantly improve the neurological function score, motor coordination ability and exploratory behavior of ICH mice, and inhibit the progression of secondary damage caused by intracranial hemorrhage from multiple dimensions. Its efficacy is superior to the positive control drug pecidadatinib (PLX3397), providing a new molecular target and interference strategy for the comprehensive treatment of ICH.

[0012] (2) Sunitinib in this invention is a drug that has been approved for clinical treatment of cancer. Its human pharmacokinetics, safety and blood-brain barrier penetration ability have sufficient clinical data to support it. This greatly reduces the development risk, cycle and cost of its indication for ICH, and has great potential to be quickly promoted to clinical application.

[0013] (3) The present invention found that short-term (3 days) sunitinib treatment during the acute phase of ICH can quickly intervene in microglial cell function, avoiding the microenvironment homeostasis disruption and potential side effects that may be caused by long-term inhibition of CSF-1R, and improving treatment safety while ensuring efficacy. Attached Figure Description

[0014] Figure 1 The modified neurological deficit score (mNSS) results for mice in different treatment groups;

[0015] Wherein, ICH represents intracranial hemorrhage, 1d, 3d, 7d, 14d, and 21d represent days 1, 3, 7, 14, and 21 after ICH modeling, Sham represents the sham-operated group, ICH+Vehicle represents the ICH model group, ICH+20mg / kg Sunitinib represents the low-dose group (20mg / kg), ICH+40mg / kg Sunitinib represents the medium-dose group (40mg / kg), ICH+60mg / kg Sunitinib represents the high-dose group (60mg / kg), ICH+40mg / kg PLX3397 represents the ICH+positive control pecidadatinib group (40mg / kg), ## indicates a significant difference compared to the sham-operated group (p < 0.01), * indicates a significant difference compared to the ICH model group (p < 0.05), and the same applies below;

[0016] Figure 2 The results of the turning test for mice in different treatment groups;

[0017] Where Cornertest(%) represents the corner test, ** indicates a significant difference compared to the ICH model group, p < 0.01, the same applies below;

[0018] Figure 3 The results of the cylinder test for mice in different treatment groups;

[0019] Where Cylindertest(%) represents the cylinder test;

[0020] Figure 4 Open field test results for mice in different treatment groups;

[0021] Figure A shows the movement trajectories of mice in different treatment groups within the open field box; Figure B shows the statistical results of the total movement distance of mice in different treatment groups; SUN represents sunitinib, PLX represents percidatinib, and Distance (m) represents the total movement distance (meters); the same applies below.

[0022] Figure 5 Iba1 in mice under different treatment groups + Cytoskeletonization analysis results;

[0023] Figure A is Iba1 + Confocal microscopy imaging results of cells, Figure B shows Iba1. + Cytoskeleton branch number analysis results, Figure C shows Iba1 +Cytoskeleton branch length analysis results; No. of branch indicates the number of cytoskeleton branches, Branch length indicates the length of cytoskeleton branches, # indicates a significant difference compared with the sham-operated group, p < 0.05; the same applies below;

[0024] Figure 6 CD86 in mice under different treatment groups + Iba1 + / Iba1 + and Arg-1 + Iba1 + / Iba1 + Results of cell proportion analysis;

[0025] Figure A shows CD86 around the hematoma. + and Iba1 + Representative immunofluorescence images, B image shows CD86. + Iba1 + / Iba1 + Analysis results of cell proportion, Figure C shows Arg-1 cells around the hematoma. + and Iba1 + Representative immunofluorescence images, D image is Arg-1 + Iba1 + / Iba1 + Results of cell proportion analysis;

[0026] Figure 7 Results of myelin sheath analysis in striatal tissue surrounding hematoma in mice from different treatment groups;

[0027] Figure A shows the results of myelin observation using transmission electron microscopy, and Figure B shows the calculated G-ratio of myelin; G radio (fold of Sham) represents the G value (relative to the sham surgery group).

[0028] Figure 8 Results of differentially expressed genes in BV2 microglia of mice in different treatment groups;

[0029] Figure A shows the KEGG functional annotation of sunitinib-related DEGs; Figure B shows the functional annotation of sunitinib-related DEGs in lipid metabolism, signal transduction, transport and catabolic metabolism, and the immune system; Figure C shows the KEGG functional annotation of siCSF-1R-related DEGs; Figure D shows the functional annotation of siCSF-1R-related DEGs in lipid metabolism, signal transduction, transport and catabolic metabolism, and the immune system; Figure E shows protein interaction analysis showing that CSF-1R / PI3K / Akt is the core gene of sunitinib-related DEGs; and Figure F shows protein interaction analysis showing that CSF-1R / PI3K / Akt is the core gene of siRNA-related DEGs.

[0030] Figure 9 Western blotting analysis results of proteins in BV2 microglia of mice in different treatment groups;

[0031] Figure A shows the Western blotting analysis results of p-Akt, Akt, and β-actin proteins in cells; Figure B shows the relative level analysis results of p-Akt protein; Figure C shows the Western blotting analysis results of p-NF-κB, NF-κB, and β-actin proteins in cells; Figure D shows the relative level analysis results of p-NF-κB protein; Hb represents hemoglobin, Relative Units of p-Akt (fold of control) represents the relative level of p-Akt protein, and Relative Units of p-NF-κB (fold of control) represents the relative level of p-NF-κB protein; the same applies below.

[0032] Figure 10 Results of high-content imaging system analysis of BV2 microglia from mice in different treatment groups;

[0033] Figure A shows the high-content imaging results of microglia, and Figure B shows the area analysis results of borodipyrrole staining regions; Area of ​​Bodipy / cell (fold of Control) represents the area of ​​borodipyrrole staining regions in a single cell (relative to the control group).

[0034] Figure 11 The results of phagocytic capacity analysis of BV2 microglia in mice from different treatment groups;

[0035] Figure A shows the high-content imaging results after microglia phagocytose fluorescent beads, and Figure B shows the analysis of the number of fluorescent beads phagocytosed by cells; Beads / cell (fold of control) represents the number of fluorescent beads in a single cell (relative to the control group).

[0036] Figure 12 ELISA analysis results of inflammatory factors in BV2 microglia of mice in different treatment groups;

[0037] Figure A shows the ELISA analysis results of the inflammatory factor IL-1β, and Figure B shows the ELISA analysis results of the inflammatory factor TNF-α. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1: Neuroprotective effect of sunitinib in a mouse model of cerebral hemorrhage

[0040] 1. Establishment of experimental animals and models

[0041] A mouse model of spontaneous intracerebral hemorrhage, closely resembling the pathological changes of human spontaneous intracerebral hemorrhage, was constructed using collagenase IV (purchased from Sigma) via stereotactic injection. The construction process was as follows:

[0042] Healthy male C57BL / 6 mice (weighing 20-25g) were randomly divided into the following 6 groups (n=8): sham operation group, ICH model group, ICH + sunitinib low-dose group (20mg / kg, purchased from LC LABS, catalog number: LCL-S-8803), ICH + sunitinib medium-dose group (40mg / kg), ICH + sunitinib high-dose group (60mg / kg), and ICH + positive control pecidadatinib group (40mg / kg, purchased from MCE, catalog number: HY-16749).

[0043] Mice in each group were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) and fixed on a stereotaxic apparatus. An incision was made along the midline of the skull, and a hole was drilled 0.4 mm posterior to the anterior fontanelle and 1.8 mm to the right of the midline. Using a microsyringe, 0.1 U of type IV collagenase (dissolved in 0.5 μL of physiological saline) was injected into the basal ganglia region (4.0 mm depth) at a rate of 0.5 μL / min. The sham-operated group received only an equal volume of physiological saline. The needle was left in place for 5 minutes and then slowly withdrawn, and the wound was sutured.

[0044] 2. Dosing regimen

[0045] Sunitinib and pericidatinib were dissolved in physiological saline containing 1% DMSO, and administered intraperitoneally to mice in all treatment groups one hour after successful ICH modeling, followed by once daily for three consecutive days; the model group and sham-operated group were given an equal volume of solvent (i.e., physiological saline containing 1% DMSO).

[0046] 3. Neurological function assessment

[0047] 3.1 Modified Neurological Deficit Scoring

[0048] Modified neurological deficit score (mNSS) was performed before modeling and on days 1, 3, 7, 14, and 21 after modeling to assess motor, sensory, reflex, and balance functions in each group of mice; results are as follows. Figure 1 As shown.

[0049] from Figure 1As can be seen, compared with the sham-operated group, the ICH model group mice exhibited severe neurological deficits with significantly elevated mNSS scores. However, after treatment with sunitinib and pericidatinib, the neurological deficits in each treatment group were repaired to varying degrees in a dose-dependent manner. In particular, the treatment effect of 60 mg / kg sunitinib was the best. At different time points, the mNSS scores of the ICH + high-dose sunitinib group mice showed significant differences from those of the ICH model group, and were close to or even better than those of the positive control pericidatinib group.

[0050] 3.2 Corner Test

[0051] On days 7, 14, and 21 after modeling, mice in each group were placed between two plates at a 30° angle and allowed to turn freely. This was repeated 10 times, and the percentage of mice turning to the right (i.e., turning to the damaged side) was recorded. The results are as follows: Figure 2 As shown.

[0052] from Figure 2 It can be seen that, compared with the sham-operated group, the percentage of right-turning mice in the ICH model group was significantly reduced, indicating severe nerve damage; however, after treatment with sunitinib or pecidadatinib, the percentage of right-turning mice in each treatment group increased, and this also showed a dose-dependent effect.

[0053] 3.3 Cylinder Test

[0054] On days 7, 14, and 21 after modeling, mice in each group were placed in transparent cylinders (8 cm in diameter and 25 cm in height). The first 10 instances of their forelimbs touching the cylinder wall during vertical exploration were recorded, and the asymmetry index was calculated as: [number of times the left forelimb touched the wall] / [number of times the left and right forelimbs touched the wall together]. Results are as follows: Figure 3 As shown.

[0055] from Figure 3 As can be seen, compared with the sham-operated group, the asymmetry index of mice in the ICH model group was significantly reduced, indicating that their nerve damage was severe; however, the asymmetry index of each treatment group increased after treatment with sunitinib or pecidadatinib, and this also showed a dose-dependent effect.

[0056] 3.4 Open Field Test

[0057] On day 21 of modeling, mice in each group were placed in a white, opaque open field box (40×40×40cm) and allowed to explore freely for 2 minutes. The total distance traveled was recorded. Results are as follows: Figure 4 As shown.

[0058] from Figure 4It can be seen that, compared with the sham-operated group, the total movement distance of mice in the ICH model group was significantly shortened, indicating that their nerve damage was severe; however, after treatment with sunitinib or pecidadatinib, the total movement distance of each treatment group was improved, and it was also dose-dependent.

[0059] Example 2: Regulatory effect of sunitinib on microglial phenotype and myelin sheath structure in perihematoma tissue

[0060] 1. Model establishment and dosing regimen

[0061] Same as Example 1.

[0062] 2. Tissue sample collection

[0063] On the third day after modeling, mice underwent cardiac perfusion fixation with paraformaldehyde, brain was harvested to prepare frozen sections, and immunofluorescence staining was performed.

[0064] 3. Immunofluorescence staining and skeleton analysis

[0065] Microglia (Iba1), a pro-inflammatory phenotypic marker (CD86), and an anti-inflammatory / repair phenotypic marker (Arg-1) were labeled with antibodies. Imaging was performed using confocal microscopy, and CD86 was analyzed using ImageJ software. + Iba1 + / Iba1 + and Arg-1 + Iba1 + / Iba1 + The proportion of cells. Meanwhile, regarding Iba1... + Cellular skeletonization analysis was performed to quantify the number and length of branches. The results are as follows: Figure 5 and Figure 6 As shown.

[0066] from Figure 5 As can be seen, compared with the sham-operated group, the ICH model group mice showed a reduction and shortening of microglia branches around the hematoma, exhibiting a typical "amoebic" activation state; while sunitinib treatment (especially the high-dose group) significantly restored the branching morphology of microglia and restored their phagocytic capacity; furthermore, the efficacy of the medium-dose and high-dose sunitinib groups was comparable to or better than that of the positive control drug pecidatinib group.

[0067] from Figure 6 As can be seen, compared with the sham-operated group, the ICH model group mice had higher CD86 levels. + Iba1 + / Iba1 + The proportion increased significantly; while the sunitinib treatment group reduced CD86. + Iba1 + / Iba1 + The ratio increased Arg-1 + Iba1 + / Iba1 + The proportions indicate that sunitinib can transform microglia from a pro-inflammatory phenotype to a repair phenotype, and the high-dose sunitinib (60 mg / kg) is more effective than the positive control drug pecididatinib, while the medium-dose sunitinib is also as effective as or better than the positive control drug pecididatinib.

[0068] 4. Observation of myelin sheath using transmission electron microscopy (TEM)

[0069] On day 21 post-modeling, striatal tissue surrounding the hematoma in mice was harvested, fixed with glutaraldehyde and osmium tetroxide, dehydrated, embedded, and then ultrathin sections were prepared. After staining with uranyl acetate and lead citrate, the sections were observed and photographed under a HITACHI HT-7800TEM. The G-ratio (axonal inner diameter / total fiber outer diameter) of the myelin sheath was calculated using ImageJ. Results are as follows: Figure 7 As shown.

[0070] from Figure 7 The results showed that the myelin sheath structure in the ICH model group was loose, and the G-ratio was significantly higher than that in the sham surgery group. Sunitinib treatment reduced the G-ratio in a concentration-dependent manner, indicating that it can effectively alleviate myelin sheath damage caused by ICH, and the high-dose sunitinib (60 mg / kg) was more effective than the positive control drug pecidatinib.

[0071] Example 3: Study on the mechanism of sunitinib regulating microglial function through the CSF-1R / PI3K / Akt axis (in vitro validation)

[0072] 1. Cell Model and Treatment

[0073] Mouse BV2 microglia were divided into a control group, a hemoglobin (Hb, 5 μM) treatment group, an Hb + sunitinib (1 μM) group, an Hb + sunitinib (3 μM) group, an Hb + sunitinib (10 μM) group, an Hb + pecidadatinib (3 μM) group, and an Hb + CSF-1R siRNA (siCSF-1R) transfection group. BV2 microglia from each group were seeded into DMEM containing 10% FBS and 1% penicillin / streptomycin and incubated at 37°C in a CO2 environment below 5%. Hb treatment was used to simulate ICH conditions for subsequent experiments.

[0074] The nucleotide sequence of the CSF-1R siRNA used in this embodiment is as follows:

[0075] Sense strand: 5′-GCACCGAAGAACAUAUACATT-3′ (SEQ ID No. 1); Antisense strand: 5′-UGUAUAUGUUCUUCGGUGCTT-3′ (SEQ ID No. 2).

[0076] 2. RNA-seq and pathway analysis

[0077] Total RNA was extracted from the hemoglobin-treated group, the Hb+sunitinib (10 μM) group, and the Hb+siCSF-1R transfection group for transcriptome sequencing, and differentially expressed genes (DEGs) analysis was performed. The results are as follows: Figure 8 As shown.

[0078] from Figure 8 As can be seen, compared with the hemoglobin-treated group, the DEGs in both the Hb+sunitinib (10 μM) group and the siCSF-1R transfection group were significantly enriched in pathways related to PI3K / Akt signaling, lipid metabolism, phagosomes, and inflammatory responses. Protein-protein interaction (PPI) network analysis identified Akt as a core node gene in both groups.

[0079] 3. Western Blotting Validation

[0080] Cellular proteins were extracted from the control group, the hemoglobin (Hb, 5 μM) treatment group, the Hb + sunitinib (1 μM) group, the Hb + sunitinib (3 μM) group, the Hb + sunitinib (10 μM) group, and the Hb + pecidatinib (3 μM) group, and Western blotting analysis was performed. The results are as follows: Figure 9 As shown.

[0081] The results showed that, compared with the control group, Hb treatment significantly increased the levels of phosphorylated Akt (p-Akt) and phosphorylated NF-κBp65 (p-p65). Both sunitinib and pericidatinib effectively inhibited this phosphorylation activation.

[0082] 4. Functional phenotypic testing

[0083] Cells from the control group, hemoglobin (Hb, 5 μM) treatment group, Hb + sunitinib (1 μM) group, Hb + sunitinib (3 μM) group, Hb + sunitinib (10 μM) group, and Hb + pecidatinib (3 μM) group were subjected to the following tests:

[0084] 4.1 Lipid droplet accumulation

[0085] Cells were stained with BODIPY 493 / 503 dye and analyzed using a high-content imaging system. The results are as follows: Figure 10As shown.

[0086] from Figure 10 It can be seen that Hb induced significant accumulation of lipid droplets in BV2 cells, and treatment with all concentrations of sunitinib significantly reduced the lipid droplet area. Among them, the efficacy of high-dose (10 μM) sunitinib was better than that of the positive control drug pecidadatinib group, and the efficacy of medium-dose (3 μM) sunitinib was comparable to that of the positive control drug pecidadatinib group.

[0087] 4.2 Phagocytic function

[0088] Cells were co-incubated with fluorescent latex beads for 2 hours, and their phagocytic ability was assessed. Results are as follows: Figure 11 As shown.

[0089] from Figure 11 As can be seen, Hb treatment significantly inhibited the phagocytic function of BV2 cells, while sunitinib treatment effectively restored their phagocytic ability. The efficacy of high-dose (10 μM) sunitinib was superior to that of the positive control drug pecidadatinib, and the efficacy of medium-dose (3 μM) sunitinib was comparable to that of the positive control drug pecidadatinib.

[0090] 4.3 Release of inflammatory factors

[0091] The levels of IL-1β and TNF-α in cell supernatant were detected by ELISA, and the results are as follows: Figure 12 As shown.

[0092] from Figure 12 It can be seen that Hb stimulation significantly increased the release levels of IL-1β and TNF-α, while sunitinib treatment significantly inhibited the production of these pro-inflammatory factors. The high-dose (10 μM) sunitinib group was more effective than the positive control drug pecidadatinib group, and the medium-dose (3 μM) sunitinib group was also comparable to the positive control drug pecidadatinib group.

[0093] The experimental results of Examples 1-3 above collectively demonstrate that the short-term sunitinib treatment regimen provided by this invention can remodel microglial function, reduce neuroinflammation and myelin damage, and ultimately significantly improve neurological prognosis by inhibiting the CSF-1R / PI3K / Akt signaling axis of microglia during the acute phase of cerebral hemorrhage; it provides a novel strategy with high clinical translational potential for the clinical treatment of ICH.

Claims

1. Application of sunitinib in the preparation of therapeutic drugs for secondary injuries caused by intracranial hemorrhage.

2. The application as described in claim 1, characterized in that, The chemical structural formula of sunitinib is as follows:

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