Use of mac-1 agonists in the preparation of medicaments for the treatment and / or amelioration of cerebral hemorrhage

Abstract

The application discloses application of a MAC-1 agonist in preparation of a medicine for treating and / or relieving cerebral hemorrhage. Specifically disclosed is the use of Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer or derivative thereof in preparation of a medicine for treating cerebral hemorrhage and secondary brain injury thereof. The medicine can inhibit the migration of immune cells across the blood-brain barrier, reduce cerebral edema, promote hematoma absorption and reduce neural inflammation by regulating the MAC-1-mediated immune adhesion signal pathway, thereby exerting a neuroprotective effect. Meanwhile, a pharmaceutical composition containing the MAC-1 agonist is provided. The application provides a new target and a drug development strategy for cerebral hemorrhage treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of MAC-1 agonists in the preparation of drugs for treating and / or alleviating cerebral hemorrhage. Background Technology

[0002] Intracerebral hemorrhage (ICH) is one of the most severe and deadliest subtypes of stroke, accounting for 10%-15% of all stroke cases. Its pathological process involves primary hematoma compression and secondary brain injury, the latter primarily driven by mechanisms such as inflammatory response, blood-brain barrier (BBB) ​​disruption, immune cell infiltration, and oxidative stress. Currently, clinical treatment options for ICH are very limited, mainly focusing on surgical hematoma evacuation, intracranial pressure reduction, hemostasis, and general supportive care. There is a lack of specific drugs that can effectively halt the progression of secondary injury and improve neurological prognosis.

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

[0004] Currently, intervention studies targeting the inflammatory response in cerebral hemorrhage largely focus on broad-spectrum anti-inflammatory drugs (such as minocycline), antioxidants (such as edaravone), or neuroprotective agents. However, these drugs often act downstream of the inflammatory pathway, with dispersed targets, limited efficacy, and may cause side effects such as infection due to systemic immunosuppression. More importantly, existing strategies generally neglect precise intervention in the early, crucial step of immune cell migration and infiltration into brain tissue. The migration of immune cells to the site of inflammation is highly dependent on the interaction between their surface adhesion molecules and 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 myeloid cells such as neutrophils, monocytes / macrophages, etc., and plays a central role in mediating cell adhesion, transendothelial migration, and phagocytic activation. Although basic research has confirmed the important role of MAC-1 in various peripheral inflammatory diseases and ischemic stroke models, no studies have yet clearly reported the core role of MAC-1 in the immune-inflammatory cascade response after cerebral hemorrhage, nor have any small molecule drugs targeting the MAC-1 pathway been developed or proposed for the treatment of cerebral hemorrhage.

[0005] Leukadherin-1 (LA-1) is a known small molecule compound that has been reported in previous studies as an allosteric regulator of MAC-1, exhibiting immunomodulatory activity in in vitro models and certain peripheral inflammatory diseases (such as arthritis). However, whether it can cross the blood-brain barrier and whether it can exert neuroprotective effects by regulating MAC-1 in a complex central nervous system disease model such as cerebral hemorrhage remains completely unknown.

[0006] In summary, existing drug treatments for cerebral hemorrhage have limitations such as imprecise targeting and downstream effects, failing to effectively block the key pathological step of immune cell migration. Therefore, this invention proposes, for the first time, targeting the MAC-1 signaling pathway and utilizing its specific small molecule regulator Leukadherin-1 (LA-1) to develop a novel drug for treating cerebral hemorrhage, aiming to regulate neuroinflammation at its source and overcome the shortcomings of existing technologies. Summary of the Invention

[0007] This invention aims to provide a novel treatment strategy that can precisely intervene in the immune inflammatory response after cerebral hemorrhage, especially for secondary brain injury, in order to solve the problems of existing technologies lacking effective drugs targeting the MAC-1 signaling pathway, as well as the problems of traditional anti-inflammatory drugs having non-concentrated targets, limited efficacy, and large side effects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides the use of a MAC-1 agonist in the preparation of a medicament for treating and / or alleviating cerebral hemorrhage, wherein the medicament is used to alleviate secondary brain injury following cerebral hemorrhage.

[0010] Preferably, the MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer, or derivative thereof.

[0011] Preferably, the treatment and / or relief of cerebral hemorrhage includes achieving one or more of the following effects through the drug: inhibiting the migration of immune cells across the blood-brain barrier, reducing cerebral edema, promoting hematoma absorption, inhibiting inflammation, improving neurological dysfunction, or protecting neurons.

[0012] Preferably, the dosage of the drug is 20 to 40 μg / g body weight.

[0013] In other embodiments, based on the commonality of the MAC-1 signaling pathway in various inflammatory diseases of the central nervous system, the drug can also be used to treat traumatic brain injury or ischemic stroke.

[0014] A second aspect of the invention provides a pharmaceutical composition for treating and / or alleviating cerebral hemorrhage.

[0015] The pharmaceutical composition comprises a therapeutically effective amount of a MAC-1 agonist and a pharmaceutically acceptable carrier.

[0016] Preferably, the MAC-1 agonist is Leukadherin-1 (LA-1) or a pharmaceutically acceptable salt, isomer, or derivative thereof.

[0017] Preferably, the pharmaceutical composition may further contain one or more other active ingredients selected from anti-inflammatory drugs, antioxidants, or neuroprotective agents to achieve a synergistic therapeutic effect.

[0018] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, injections, sustained-release formulations, and nano-formulations.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention is the first to explicitly identify MAC-1 (integrin αMβ2) as a drug target for treating cerebral hemorrhage. By using MAC-1 agonists (such as LA-1) to modulate this signaling pathway, it is possible to precisely intervene in the adhesion and migration of immune cells at the source, effectively targeting the core pathological aspects of secondary brain injury. This strategy not only synergistically achieves multiple therapeutic effects such as reducing cerebral edema, promoting hematoma absorption, inhibiting neuroinflammation, protecting neurons, and improving neurological dysfunction, but also potentially improves treatment safety by regulating immune homeostasis rather than broadly suppressing it. Furthermore, the regulatory mechanism revealed in this invention provides a novel direction for drug development in the treatment of traumatic brain injury, ischemic stroke, and other central nervous system inflammatory diseases, with broad application prospects. Attached Figure Description

[0021] Figure 1 This is a diagram showing the proteomic analysis results of the cerebral hemorrhage model in Example 1 of the present invention, revealing that ITGB2 and ITGAM are core integrin genes for immune activation; among them, Figure 1 A is a schematic diagram of the experimental procedure for a mouse model of cerebral hemorrhage and proteomics analysis. Figure 1 B is a volcano plot of differentially expressed proteins (DEPs) between the ICH group and the sham-operated group. Figure 1 C is a hierarchical clustering heatmap of protein expression patterns in the ICH group and the control group. Figure 1 D is a graph showing the enrichment of differentially expressed proteins through the KEGG pathway. Figure 1 E is a chord diagram illustrating the association 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 located at a hub position with high connectivity.

[0022] Figure 2This is a graph showing the effect of LA-1 treatment on improving neurological function and tissue damage in mice with cerebral hemorrhage in Example 2 of this invention; wherein, Figure 2 A is a schematic diagram of the experimental design and drug administration timeline of a mouse model of cerebral hemorrhage. Figure 2 B shows T2-weighted magnetic resonance imaging (MRI) images of mice in each group at different time points. Figure 2 C is a quantitative statistical graph of brain edema volume in each group of mice on day 3. Figure 2 D is a quantitative statistical graph showing the percentage reduction in hematoma volume in each group of mice on day 12. Figure 2 E shows the modified neurological deficit score (mNSS) results for each group of mice. Figure 2 F shows the results of the cornering test for each group of mice. Figure 2 G shows the results of the forelimb symmetry test for each group of mice. Figure 2 H represents the Fluoro-Jade C (FJC) and DAPI staining images of brain tissue from each group of mice, showing the degree of neuronal degeneration. Figure 2 I is a quantitative statistical graph showing the number of FJC positive neurons.

[0023] Figure 3 This is a diagram showing the experimental results of LA-1's immune regulation of the LPS-induced neuroinflammation model in Example 3 of this invention; wherein, Figure 3 A shows the MPO immunohistochemical staining images of the brain tissue of mice in each group, indicating the infiltration of neutrophils. Figure 3 B is a quantitative statistical graph showing the number of MPO-positive cells. Figure 3 C shows the IBA1 immunohistochemical staining images of the brain tissue of mice in each group, indicating the activation status of microglia / macrophages. Figure 3 D is a quantitative statistical graph showing the number of IBA1-positive cells. Figure 3 E shows the immunofluorescence staining of the cerebral cortex of mice in each group, showing the colocalization of MPO (green), CD11b (yellow) and Claudin-5 (red), and DAPI (blue) staining of cell nuclei; Figure 3 F is a statistical graph showing the IL-1β content in brain tissue as detected by ELISA. Figure 3 G is a statistical graph of TNF-α content in brain tissue detected by ELISA. Figure 3 H is a statistical graph of IL-6 levels in brain tissue detected by ELISA. Figure 3 I is a statistical graph of IL-12 levels in brain tissue detected by ELISA. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All materials and instruments used in the embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0025] The main experimental materials and reagents used in this invention are shown in Table 1.

[0026] Table 1 Main Materials and Reagents

[0027]

[0028] The main instruments and equipment used in this invention are shown in Table 2.

[0029] Table 2 Main Instruments

[0030]

[0031] Example 1: Identification of core targets of immune inflammation after cerebral hemorrhage through proteomics analysis

[0032] This invention uses proteomics analysis to study the changes in protein expression profiles in the brain tissue surrounding the hematoma after cerebral hemorrhage in mice, and to screen key molecules and signaling pathways involved in secondary injury. The specific steps are as follows:

[0033] (1) Establishment of animal models

[0034] Male C57BL / 6J mice (purchased from Charles River) aged 8-10 weeks and weighing 22–25 grams were used to establish a brain hemorrhage model using autologous blood induction. Mice were housed in a 12-hour light / dark cycle environment with free access to food and water. All experimental procedures were approved by the Ethics Committee of the Beijing Neurosurgical Institute and followed the guidelines for laboratory animal care and use.

[0035] This study established a mouse model of intracranial hemorrhage (ICH) using stereotactic injection of autologous venous blood. The specific steps were as follows: Anesthesia was induced by intraperitoneal injection of tribromoethanol, and a heating pad was used to maintain the body temperature at 37°C during the procedure. Approximately 50–100 μL of autologous venous blood was collected directly from the right orbital horn vein through a glass capillary tube into a 1 mL syringe, minimizing air exposure and disturbance during the procedure to prevent clotting. The mice were then fixed in a stereotactic frame. Before blood collection, a cranial foramen with a diameter of approximately 0.5 mm was drilled at a location 0.2 mm anterior to the anterior fontanelle and 2.5 mm to the right. Using a 26G needle, 25 μL of autologous arterial blood was slowly injected at a rate of 2.5 μL / min to a depth of 3.5 mm ventral to the anterior fontanelle. After injection, the needle was left in place for 5 minutes to prevent backflow. The cranial foramen was then sealed with bone wax, and the skin was sutured.

[0036] The sham surgery group underwent the same procedures as the other group, except that no autologous blood was injected.

[0037] (2) Sample collection

[0038] On the 5th day after modeling, brain tissue (approximately 20 mg) around the hematoma was collected.

[0039] (3) Proteomics analysis

[0040] Total protein was extracted using RIPA lysis buffer and enzymatically digested into peptides via filter-assisted sample preparation (FASP). Peptide samples were subjected to data-independent acquisition (DIA) using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Raw mass spectrometry data were processed using MaxQuant software, and proteins were identified using the UniProt mouse reference database. Label-free quantification (LFQ) was used to assess the relative protein abundance among samples.

[0041] (4) Bioinformatics analysis

[0042] Differentially expressed proteins were screened based on a |Fold Change| > 1.5 and a p-value < 0.05. KEGG pathway enrichment analysis and protein-protein interaction (PPI) network analysis were performed on the differentially expressed proteins (using the STRING database and Cytoscape software).

[0043] The results are as follows Figure 1 As shown, the volcano map and hierarchical clustering heatmap ( Figure 1 A-1B clearly demonstrated significant differences in protein expression between groups. KEGG pathway enrichment analysis ( Figure 1C-1D analysis showed that these differentially expressed proteins were significantly enriched in immune-inflammatory pathways such as leukocyte transendothelial migration, neutrophil extracellular trap (NET) formation, and phagosomes. Particularly important was the PPI network analysis (…). Figure 1 E) revealed that the integrase subunits ITGB2 (CD18) and ITGAM (CD11b) occupy a core hub position in the network with the highest connectivity, indicating that the MAC-1 complex composed of them is a core regulatory node for the immune inflammatory response after cerebral hemorrhage.

[0044] This embodiment systematically demonstrates through proteomics technology that MAC-1 (ITGAM / ITGB2) is a key target in the immune activation network after cerebral hemorrhage.

[0045] Example 2: The therapeutic effect of LA-1 on neurological function and tissue damage in mice with cerebral hemorrhage

[0046] This embodiment aims to evaluate the neuroprotective efficacy of the small molecule compound Leukadherin-1 (LA-1) in an animal model of cerebral hemorrhage. The specific steps are as follows:

[0047] (1) Animal models and grouping

[0048] A mouse model of cerebral hemorrhage was constructed according to the method described in Example 1, and the model mice were divided into three groups: cerebral hemorrhage model group (ICH group), LA-1 low-dose group (20 μg / g), and LA-1 high-dose group (40 μg / g), with n=10 in each group.

[0049] (2) Administration

[0050] 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.

[0051] (3) Magnetic resonance imaging (MRI) assessment

[0052] 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.

[0053] (4) Neurobehavioral assessment

[0054] Modified Neurological Deficit Score (mNSS): assesses motor, sensory, reflex, and balance functions; a higher score indicates a more severe deficit.

[0055] Turning angle test: Record the number of times the mouse turns to the left or right, and calculate the turning deviation ratio.

[0056] 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.

[0057] (5) Histological analysis

[0058] 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.

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] Example 3: Immunomodulatory effect of LA-1 on an LPS-induced neuroinflammation model

[0064] This embodiment aims to verify the immunomodulatory effect of LA-1 in an inflammatory nerve injury model, eliminate the interference of hematoma factors, and further confirm its anti-inflammatory mechanism. The specific steps are as follows:

[0065] (1) Model building and grouping

[0066] A mouse model of neuroinflammation was induced using lipopolysaccharide (LPS), and the mice were randomly divided into three groups: an LPS (10 μg / g) model group, a low-dose LPS+LA-1 group (20 μg / g), and a high-dose LPS+LA-1 group (40 μg / g). The neuroinflammation mouse model was established by injecting LPS using a stereotaxic instrument. The specific steps were as follows: Anesthesia was induced by intraperitoneal injection of tribromoethanol, and a heating pad was used to maintain the body temperature at 37°C during the operation. The mice were then fixed on the stereotaxic instrument. Before injection, a cranial foramen with a diameter of approximately 0.5 mm was drilled at a location 0.2 mm anterior to and 2.5 mm to the right of the anterior fontanelle. Using a 26G injection needle, 10 μL of proportionally diluted LPS (10 μg / g) was slowly injected at a rate of 1 μL / min to a depth of 3.5 mm ventrally from the anterior fontanelle. After injection, the needle was left in place for 5 minutes to prevent blood backflow. The cranial foramen was then sealed with bone wax, and the skin was sutured.

[0067] (2) Immunohistochemical detection / immunofluorescence detection

[0068] Twenty-four hours after drug administration, brain tissue was perfused and harvested. Paraffin sections were prepared, and neutrophils were labeled with anti-MPO antibody, while microglia / macrophages were labeled with anti-IBA1 antibody and quantitatively analyzed. Triple immunofluorescence staining with anti-CD11b, anti-Claudin-5 (blood-brain barrier tight junction protein), and anti-MPO antibody was performed, and the relative positions of immune cells and blood vessels were observed using confocal microscopy.

[0069] (3) Detection of inflammatory factors

[0070] Brain tissue homogenate supernatant was collected, and the concentrations of IL-1β, TNF-α, IL-6, and IL-12 were detected using an ELISA kit.

[0071] Immunohistochemical results showed ( 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 cell types in a dose-dependent manner.

[0072] Immunofluorescence results showed ( Figure 3 In the LPS group, a large number of MPO⁺ / CD11b⁺ immune cells crossed the Claudin-5 positive blood vessel walls and entered the brain parenchyma; while in the high-dose LA-1 group, the vast majority of immune cells were confined within the blood vessel lumen, indicating that LA-1 effectively maintained the integrity of the blood-brain barrier.

[0073] ELISA results ( Figure 3F-3I) indicates that LA-1 treatment can significantly reduce the levels of key pro-inflammatory factors such as IL-1β, TNF-α, IL-6 and IL-12 in brain tissue.

[0074] This embodiment demonstrates that LA-1 effectively inhibits the migration of immune cells across the blood-brain barrier and reduces central nervous system inflammatory response by regulating MAC-1 function, which provides a key mechanistic explanation for its protective role in the treatment of cerebral hemorrhage.

[0075] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of Leukadherin-1 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating and / or alleviating cerebral hemorrhage, characterized in that, The treatment and / or relief of cerebral hemorrhage refers to achieving at least one of the following effects: reducing cerebral edema, improving neurological dysfunction, and / or protecting neurons.

2. The application according to claim 1, characterized in that, The dosage of the drug is 20-40 μg / g body weight.

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