Application of MAC-1 agonist in preparation of medicine for treating and / or relieving cerebral hemorrhage

Abstract

The invention discloses application of an MAC-1 agonist in preparation of a medicine for treating and / or relieving cerebral hemorrhage. Specifically, the invention discloses application of Leukadherin-1 (LA-1) or pharmaceutically acceptable salts, isomers or derivatives thereof in preparation of medicines for treating cerebral hemorrhage and secondary brain injury thereof. The medicine can inhibit the migration of immune cells across the blood brain barrier, relieve encephaledema, promote hematoma absorption and reduce neuroinflammation by adjusting an MAC-1 mediated immune adhesion signal channel, thereby playing a role in neuroprotection. Meanwhile, the invention provides a pharmaceutical composition containing the MAC-1 agonist. The invention provides a new target and a new drug development strategy for cerebral hemorrhage treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of MAC-1 agonist in the preparation of drugs for treating and / or relieving cerebral hemorrhage. BACKGROUND

[0002] Cerebral hemorrhage (ICH) is one of the most severe and highest mortality subtypes of stroke, accounting for 10-15% of all stroke cases. Its pathological process includes primary hematoma compression and secondary brain injury, which is mainly driven by mechanisms such as inflammatory response, blood brain barrier (BBB) disruption, immune cell infiltration and oxidative stress. At present, the clinical treatment of cerebral hemorrhage is very limited, mainly including surgical hematoma removal, reduction of intracranial pressure, hemostasis and general supportive treatment, and there is still a lack of specific drugs that can effectively block the progression of secondary injury and improve the prognosis of neurological function.

[0003] Among the many mechanisms of secondary injury after cerebral hemorrhage, immune inflammatory response is considered as the core link. Hematoma components and their degradation products can activate microglia cells in the central nervous system and recruit peripheral neutrophils, monocytes / macrophages, etc. These activated immune cells release a large amount of pro-inflammatory cytokines (such as IL-1β, TNF-α, IL-6, etc.) and reactive oxygen species, further exacerbating the permeability of the blood brain barrier, leading to worsening brain edema and triggering neuronal apoptosis and necrosis. Therefore, targeting the regulation of neuroinflammation has become a promising strategy for the treatment of cerebral hemorrhage.

[0004] At present, the intervention research on inflammatory response of cerebral hemorrhage is mostly focused on broad-spectrum anti-inflammatory drugs (such as minocycline), antioxidants (such as edaravone) or neuroprotective agents. However, these drugs often act on the downstream of the inflammatory pathway, with scattered targets, limited efficacy, and possible side effects such as infection due to systemic immunosuppression. More importantly, the existing strategies generally ignore the precise intervention of the early key step of immune cell migration and infiltration into brain tissue. The migration of immune cells to the inflammatory site is highly dependent on the interaction between the adhesion molecules on their surface and the vascular endothelial cells. Among them, Macrophage-1 antigen (MAC-1, i.e. integrin αMβ2, composed of CD11b and CD18 subunits) is a key integrin expressed on the surface of neutrophils, monocytes / macrophages and other myeloid cells, which plays a central role in mediating cell adhesion, transendothelial migration and phagocytic activation. Although basic research has confirmed the importance of MAC-1 in various peripheral inflammatory diseases and ischemic stroke models, there is no research to report the core position of MAC-1 in the immune inflammatory cascade reaction after cerebral hemorrhage, and there is no small molecule drug targeting MAC-1 pathway developed or proposed for the treatment of cerebral hemorrhage.

[0005] Leukadherin-1 (LA-1) is a known small molecule compound, which has been reported as an allosteric modulator of MAC-1 in previous studies, and shows immunomodulatory activity in in vitro models and certain peripheral inflammatory diseases (such as arthritis). However, it is completely unknown whether it can penetrate the blood-brain barrier and whether it can play a neuroprotective role by regulating MAC-1 in the complex central nervous system disease model of intracerebral hemorrhage.

[0006] In summary, the existing drug treatment for intracerebral hemorrhage has limitations such as inaccurate target and downstream effect, and cannot effectively block the key pathological link of immune cell migration. Therefore, the present application first proposes to target the MAC-1 signaling pathway and use the specific small molecule modulator Leukadherin-1 (LA-1) to develop a new drug for treating intracerebral hemorrhage, aiming to regulate neuroinflammation from the source and solve the problems of the prior art. SUMMARY

[0007] The present application aims to provide a new treatment strategy that can accurately intervene in the immune inflammatory response after intracerebral hemorrhage, especially for secondary brain injury, to solve the problems of the prior art such as the lack of effective drugs targeting the MAC-1 signaling pathway, and the non-central target, limited efficacy and large side effects of traditional anti-inflammatory drugs.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The first aspect of the present application provides a use of a MAC-1 agonist in the preparation of a medicament for treating and / or alleviating intracerebral hemorrhage, wherein the medicament is used to alleviate secondary brain injury after intracerebral hemorrhage.

[0009] Preferably, the MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer or derivative thereof.

[0010] Preferably, the treatment and / or alleviation of intracerebral hemorrhage includes one or more of the following effects achieved by the medicament: inhibition of immune cell migration across the blood-brain barrier, reduction of brain edema, promotion of hematoma absorption, inhibition of inflammation, improvement of neurological dysfunction, or protection of neurons.

[0011] Preferably, the administration dose of the medicament is 20 to 40 μg / g of body weight.

[0012] In other embodiments, based on the commonality of the MAC-1 signaling pathway in various central nervous system inflammatory diseases, the medicament can also be used to treat brain trauma or ischemic stroke.

[0013] The second aspect of the present application provides a pharmaceutical composition for treating and / or alleviating intracerebral hemorrhage.

[0014] The pharmaceutical composition comprises a therapeutically effective amount of the MAC-1 agonist and a pharmaceutically acceptable carrier.

[0015] Preferably, the MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer or derivative thereof.

[0016] Preferably, the pharmaceutical composition can further comprise one or more other active ingredients selected from anti-inflammatory agents, antioxidants or neuroprotective agents to achieve a synergistic therapeutic effect.

[0017] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, injections, sustained-release preparations, nano-preparations, etc.

[0018] The beneficial effects of the present application are as follows: The present application first identifies MAC-1 (integrin alpha M beta 2) as a drug action target for treating cerebral hemorrhage, and by using a MAC-1 agonist (such as LA-1) to regulate the signaling pathway, the adhesion and migration of immune cells can be precisely intervened from the source, effectively targeting the core pathological link of secondary brain injury. This strategy not only synergistically achieves multiple therapeutic effects of reducing brain edema, promoting hematoma absorption, inhibiting neuroinflammation, protecting neurons and improving neurological dysfunction, but also potentially improves treatment safety by regulating immune homeostasis rather than extensive inhibition. In addition, the regulatory mechanism disclosed in the present application provides a new direction for drug development for the treatment of other central nervous inflammatory diseases such as brain trauma and ischemic stroke, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure A is a schematic diagram of the experimental process for the cerebral hemorrhage model and proteomics analysis of mice in Example 1 of the present application, which reveals that ITGB2 and ITGAM are core integrin genes for immune activation. Figure 1 A is a schematic diagram of the experimental process for the cerebral hemorrhage model and proteomics analysis of mice in Example 1 of the present application, which reveals that ITGB2 and ITGAM are core integrin genes for immune activation. Figure 1 B is a volcano plot of differentially expressed proteins (DEPs) between the ICH group and the sham operation group, Figure 1 C is a hierarchical clustering heat map of protein expression patterns in ICH group and control group samples, Figure 1 D is a KEGG pathway enrichment analysis diagram of differentially expressed proteins, Figure 1 E is a chord diagram showing the correlation between differentially expressed proteins and key immune pathways, Figure 1 F is a protein-protein interaction network (PPI) analysis diagram showing that ITGB2 and ITGAM are in a high-connectivity hub position.

[0020] Figure 2 Figure A is a schematic diagram of the experimental process for the cerebral hemorrhage model and proteomics analysis of mice in Example 1 of the present application, which reveals that ITGB2 and ITGAM are core integrin genes for immune activation.Figure 2 A is the experimental design and drug administration time line of the brain hemorrhage mouse model, Figure 2 B is the T2-weighted magnetic resonance imaging (MRI) image of each group of mice at different time points, Figure 2 C is a quantitative statistical graph of brain edema volume of each group of mice on day 3, Figure 2 D is a quantitative statistical graph of the percentage of blood volume reduction of each group of mice on day 12, Figure 2 E is a result graph of the modified neurological severity score (mNSS) of each group of mice, Figure 2 F is a result graph of the turning test of each group of mice, Figure 2 G is a result graph of the forelimb use symmetry test of each group of mice, Figure 2 H is a Fluoro-Jade C (FJC) and DAPI staining graph of the brain tissue of each group of mice, showing the neuronal degeneration, Figure 2 I is a quantitative statistical graph of the number of FJC positive neurons.

[0021] Figure 3 is the experimental result graph of the immune regulation of LA-1 on the LPS-induced neuroinflammation model in Example 3 of the present application; wherein, Figure 3 A is a MPO immunohistochemical staining graph of the brain tissue of each group of mice, showing the neutrophil infiltration, Figure 3 B is a quantitative statistical graph of the number of MPO positive cells, Figure 3 C is an IBA1 immunohistochemical staining graph of the brain tissue of each group of mice, showing the activation of microglia / macrophages, Figure 3 D is a quantitative statistical graph of the number of IBA1 positive cells, Figure 3 E is an immunofluorescence staining graph of the cerebral cortex of each group of mice, showing the colocalization of MPO (green), CD11b (yellow) and Claudin-5 (red), and DAPI (blue) for nuclear staining; Figure 3 F is a statistical graph of the content of IL-1β in the brain tissue detected by ELISA, Figure 3 G is a statistical graph of the content of TNF-α in the brain tissue detected by ELISA, Figure 3 H is a statistical graph of the content of IL-6 in the brain tissue detected by ELISA, Figure 3 I is a statistical graph of the content of IL-12 in the brain tissue detected by ELISA. DETAILED DESCRIPTION

[0022] The present application will be further described in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The materials and instruments used in the examples can be purchased through commercial channels. The experimental methods not specified in the examples are usually carried out under conventional conditions or under the conditions recommended by the manufacturer.

[0023] The main experimental materials and reagents used in the present application are shown in Table 1.

[0024] Table 1 Main materials and reagents

[0025] The main instruments and equipment used in the present application are shown in Table 2.

[0026] Table 2 Main instruments

[0027] Example 1 Identification of core target points of immune inflammation after cerebral hemorrhage by proteomics analysis The present application studies the protein expression profile changes of the brain tissue around the hematoma after cerebral hemorrhage in mice by proteomics analysis, and screens the key molecules and signal pathways involved in secondary injury. The specific steps are as follows: (1) Establishment of animal model 8-10 week old, 22-25 g male C57BL / 6J mice (purchased from Charles River) were selected to establish a cerebral hemorrhage model by autologous blood induction. The mice were raised in a 12-hour light / dark cycle environment and had free access to food and water. All experimental procedures were approved by the Ethics Committee of Beijing Institute of Neurosurgery and followed the guidelines for the care and use of experimental animals.

[0028] In this study, a mouse intracerebral hemorrhage (ICH) model was established by stereotactic injection of autologous venous blood. The specific steps are as follows: anesthesia was induced by intraperitoneal injection of tribromoethanol, and a heating pad was used to maintain body temperature at 37°C during the operation. About 50-100 μL of autologous venous blood was collected from the right orbital venous angle into a 1 mL syringe through a glass capillary tube, and air exposure and disturbance were minimized during the operation to prevent blood clotting. Then the mice were fixed on the stereotactic instrument. Before blood collection, a skull hole with a diameter of about 0.5 mm was drilled at a distance of 0.2 mm from the front point and 2.5 mm from the right side. Using a 26G injection needle, 25 μL of autologous arterial blood was slowly injected at a speed of 2.5 μL / min to a depth of 3.5 mm from the ventral side of the front point. After the injection was completed, the needle was left in place for 5 minutes to prevent blood reflux. Then bone wax was used to seal the skull hole, and the skin was sutured.

[0029] The sham operation group was the same as the operation group except that no autologous blood was injected.

[0030] (2) Sample collection On the 5th day after modeling, the brain tissue around the hematoma (about 20 mg) was taken.

[0031] (3) Proteomics analysis Total proteins were extracted using RIPA lysis buffer and digested into peptides by filter-aided sample preparation (FASP). Peptide samples were subjected to data-independent acquisition (DIA) by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Raw mass spectrometry data were processed using MaxQuant software and protein identification was performed by UniProt mouse reference database. Relative protein abundance among samples was evaluated by label-free quantification (LFQ) method.

[0032] (4) Bioinformatics analysis Differentially expressed proteins were screened according to the criteria of |Fold Change|>1.5 and p-value<0.05. KEGG pathway enrichment analysis and protein-protein interaction (PPI) network analysis were performed on the differential proteins (using STRING database and Cytoscape software).

[0033] The results are shown in Figure 1 Volcano plots and hierarchical clustering heatmaps Figure 1 A-1B) clearly showed the significant differences in protein expression among groups. KEGG pathway enrichment analysis Figure 1 C-1D) showed that these differential proteins were significantly enriched in immune inflammation-related pathways such as "leukocyte transendothelial migration", "neutrophil extracellular trap (NET) formation", and "phagosome". Most importantly, PPI network analysis Figure 1 E) revealed that integrin subunits ITGB2 (CD18) and ITGAM (CD11b) were in the core hub position of the network with the highest connectivity, indicating that the MAC-1 complex composed of them was the core regulatory node of immune inflammatory response after intracerebral hemorrhage.

[0034] This example systematically proves that MAC-1 (ITGAM / ITGB2) is a key target in the immune activation network after intracerebral hemorrhage by proteomics technology.

[0035] Example 2 LA-1 on the therapeutic effect of nerve function and tissue damage in mice with intracerebral hemorrhage This example aims to evaluate the neuroprotective efficacy of the small molecule compound Leukadherin-1 (LA-1) in an animal model of intracerebral hemorrhage. The specific steps are as follows: (1) Animal model and grouping According to the method described in Example 1, an intracerebral hemorrhage mouse model was constructed, and the model mice were divided into 3 groups: intracerebral hemorrhage model group (ICH group), LA-1 low-dose group (20 μg / g), and LA-1 high-dose group (40 μg / g), n=10 in each group.

[0036] (2) Drug administration One hour after successful model establishment, LA-1 was administered via intraperitoneal injection (the ICH group received an equal volume of solvent, 200 μL), twice daily for 12 days.

[0037] (3) Magnetic resonance imaging (MRI) assessment On days 3 and 12 post-modeling, T2-weighted imaging was performed using 7.0T small animal MRI to measure cerebral edema volume and residual hematoma volume.

[0038] (4) Neurobehavioral assessment Modified Neurological Deficit Score (mNSS): assesses motor, sensory, reflex, and balance functions; a higher score indicates a more severe deficit.

[0039] Turning angle test: Record the number of times the mouse turns to the left or right, and calculate the turning deviation ratio.

[0040] Forelimb use asymmetry test: The number of times mice used their left, right, or both forelimbs to support their bodies during exploration was recorded in a transparent cylinder, and the symmetry score was calculated.

[0041] (5) Histological analysis At the end of the experiment, brain tissue was taken for frozen sections, and degenerated neurons were stained with Fluoro-Jade C (FJC) and counterstained with DAPI. The number of FJC-positive cells around the hematoma was counted under a fluorescence microscope.

[0042] MRI results showed ( Figure 2 Compared with the model group, the high-dose LA-1 group showed a significant reduction in cerebral edema volume on day 3 and a significant increase in hematoma absorption rate on day 12.

[0043] Behavioral results showed that the mNSS score was significantly reduced in both the high-dose and low-dose LA-1 groups. Figure 2 E), the abnormal turning behavior in the turning angle experiment was corrected ( Figure 2 F), forelimb symmetry was significantly improved ( Figure 2 G).

[0044] Histological results: FJC staining showed ( Figure 2 In the H-2I model group, there were a large number of FJC-positive degenerated neurons around the hematoma, while in the LA-1 treatment group, especially the high-dose group, the number of positive cells was significantly reduced.

[0045] This embodiment demonstrates that LA-1 can significantly reduce cerebral edema after cerebral hemorrhage, promote hematoma absorption, reduce neuronal degeneration, and effectively improve neurological function recovery, exhibiting a clear neuroprotective effect.

[0046] Example 3: Immunomodulatory effect of LA-1 on an LPS-induced neuroinflammation model This example aims to verify the immunomodulatory effect of LA-1 in an inflammatory nerve injury model, excluding the interference of hematoma factors, and further confirming its anti-inflammatory mechanism. The specific steps are as follows: (1) Model establishment and grouping A lipopolysaccharide (LPS) induced nerve inflammation mouse model was used, and the model mice were randomly divided into: LPS (10 μg / g) model group, LPS+LA-1 low dose group (20 μg / g), LPS+LA-1 high dose group (40 μg / g). The nerve inflammation mouse model was established by stereotaxic apparatus. Anesthesia was induced by intraperitoneal injection of tribromoethanol, and a heating pad was used to maintain body temperature at 37°C during the operation. Then the mice were fixed on the stereotaxic apparatus. Before injection, a skull hole with a diameter of about 0.5 mm was drilled at a distance of 0.2 mm from the front point and 2.5 mm from the right side. Using a 26G injection needle, 10 μL of proportionally diluted LPS (10 μg / g) was slowly injected at a speed of 1 μL / min to a depth of 3.5 mm from the ventral side of the front point. After injection, the needle was left in place for 5 minutes to prevent blood reflux. Then the skull hole was sealed with bone wax, and the skin was sutured.

[0047] (2) Immunohistochemical detection / immunofluorescence detection The brain was perfused 24 hours after administration, paraffin section, and labeled with anti-MPO antibody to mark neutrophils and anti-IBA1 antibody to mark microglia / macrophages, and quantitative analysis was performed. Triple immunofluorescence staining was performed using anti-CD11b, anti-Claudin-5 (blood-brain barrier tight junction protein) and anti-MPO antibodies, and the relative position of immune cells and blood vessels was observed by confocal microscopy.

[0048] (3) Inflammatory factor detection The brain tissue homogenate supernatant was detected by ELISA kit to detect the concentration of IL-1β, TNF-α, IL-6 and IL-12.

[0049] The results of immunohistochemical detection showed that Figure 3 A-3D), LPS stimulation led to a sharp increase in the number of MPO⁺ neutrophils and IBA1⁺ cells in the brain, while LA-1 treatment significantly reduced the infiltration of these two types of cells in a dose-dependent manner.

[0050] The results of immunofluorescence showed that Figure 3 E), in the LPS group, a large number of MPO⁺ / CD11b⁺ immune cells crossed the Claudin-5 positive blood vessel wall into the brain parenchyma; while in the LA-1 high dose group, most of the immune cells were confined to the blood vessel lumen, indicating that LA-1 effectively maintained the integrity of the blood-brain barrier.

[0051] ELISA results (Figure 3I) showed that LA-1 treatment significantly reduced the levels of key proinflammatory cytokines, including IL-1 β, TNF-α, IL-6, and IL-12 in brain tissues. Figure 3

[0052] This example demonstrates that LA-1 effectively suppresses the transmigration of immune cells across the blood-brain barrier by modulating MAC-1 function, and attenuates central nervous inflammation, which provides a key mechanistic explanation for its protective role in the treatment of intracerebral hemorrhage.

[0053] Although the embodiments of the present application have been shown and described above, it should be understood by those having ordinary skill in the art that the above embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations of the above embodiments can be made within the scope of the present application.​

Claims

1. Use of a MAC-1 agonist in the preparation of a medicament for treating and / or alleviating cerebral hemorrhage, the medicament being used to alleviate secondary brain injury after cerebral hemorrhage.

2. Use according to claim 1, characterized in that, The MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer or derivative thereof.

3. Use according to claim 1, characterized in that, The treatment and / or alleviation of cerebral hemorrhage comprises one or more of inhibiting the migration of immune cells across the blood-brain barrier, reducing brain edema, promoting hematoma absorption, inhibiting inflammation, improving neurological dysfunction, or protecting neurons.

4. Use according to claim 1, characterized in that, The medicament is administered at a dose of 20-40 μg / g body weight.

5. The use according to claim 1, characterized in that, The medicament is also used to treat brain trauma or ischemic stroke.

6. A pharmaceutical composition for treating and / or alleviating cerebral hemorrhage, characterized by, The medicament comprises a therapeutically effective amount of a MAC-1 agonist and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein, The MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer or derivative thereof.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that, The pharmaceutical composition further comprises one or more drugs selected from anti-inflammatory drugs, antioxidants or neuroprotective agents.

9. The pharmaceutical composition of claim 6, wherein, The dosage form of the pharmaceutical composition comprises injections, sustained-release preparations, nano-preparations.

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