Use of alvelestat in the manufacture of a medicament for treating intracerebral hemorrhage

Abstract

The application discloses application of Alvelestat in preparation of a medicine for treating cerebral hemorrhage. The application is based on proteomic stratification analysis of hematoma fluid of a cerebral hemorrhage patient, finds that neutrophil elastase (NE) is a key molecule in the progression of cerebral hemorrhage, and further proposes a technical scheme of taking NE as a target and using a selective NE inhibitor Alvelestat for intervention. Animal experiments verify that Alvelestat can reduce cerebral edema, promote hematoma absorption, reduce neuron apoptosis and improve neural function defects; transcriptomics reveals that it plays a protective role by inhibiting multiple inflammation and immunity related signal pathways. The application provides a new strategy for precise targeted treatment of cerebral hemorrhage, and has clear clinical conversion value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the novel use of Alvelestat in the preparation of drugs for the treatment of cerebral hemorrhage. Background Technology

[0002] Intracerebral hemorrhage (ICH) is one of the most serious types of stroke, with high mortality and disability rates. Current clinical treatment primarily focuses on symptomatic support and surgical intervention, including measures such as reducing intracranial pressure, controlling cerebral edema, and, when necessary, removing the hematoma. While these treatments can alleviate the primary injury to some extent, they cannot effectively prevent the occurrence and progression of secondary brain injury following ICH at the molecular level. Secondary injury after ICH involves complex pathological processes such as activated inflammatory responses, peripheral immune cell infiltration, blood-brain barrier disruption, increased oxidative stress, cerebral edema formation, and neuronal death. This process is a key factor determining patient prognosis. To date, no universally recognized specific drugs have been approved for use that can significantly improve the prognosis of ICH.

[0003] In the field of drug development for cerebral hemorrhage, existing technologies generally focus on the regulation of inflammatory responses. Existing drug intervention strategies mainly include the following categories: anti-inflammatory pathway intervention, i.e., inhibiting pro-inflammatory cytokines or inflammatory signaling pathways such as NF-κB; antioxidant strategies, i.e., using free radical scavengers or Nrf2 pathway activators; immune cell suppression strategies, i.e., inhibiting microglia activation or reducing peripheral leukocyte infiltration into brain tissue; and gene or RNA intervention strategies, i.e., regulating inflammation-related signaling pathways such as MAPK and PI3K / Akt. However, most of these strategies focus on downstream aspects of the inflammatory response, mainly targeting processes such as the release of inflammatory mediators, oxidative stress damage, or glial cell activation. They typically only alleviate some of the consequences of secondary damage and lack direct intervention on key initiation steps such as early recruitment, adhesion, migration, and infiltration across the blood-brain barrier of immune cells.

[0004] In the immune-inflammatory response following cerebral hemorrhage, neutrophils are among the first peripheral immune cells to infiltrate the lesion area. Neutrophils participate in secondary brain injury through degranulation, release of inflammatory mediators, oxidative stress generation, and the formation of neutrophil extracellular traps (NETs). The NET is a network structure composed of an extracellular DNA backbone, histones, and various neutrophil granule proteins, including key components such as myeloperoxidase (MPO) and neutrophil elastase (NE). Current technology indicates that neutrophil elastase is not only an important component of the NET but also directly participates in its formation.

[0005] In recent years, inhibitors targeting neutrophil elastase have attracted attention in the field of brain injury. Existing technologies include methods for using the neutrophil elastase inhibitor Sivelestat in animal models of cerebral ischemia, and attempts to use it in animal models of cerebral hemorrhage. However, these existing technologies have the following limitations: First, research on Sivelestat mainly focuses on cerebral ischemia, and its application in cerebral hemorrhage is insufficient, with its mechanism of action remaining unclear. Second, Sivelestat is administered intravenously, limiting its application in pre-hospital emergency care or long-term treatment. Third, existing technologies generally rely on directly validating interventions based on known targets, lacking a complete technical pathway for systematically screening and validating key targets from clinical samples of cerebral hemorrhage patients.

[0006] On the other hand, Alvelestat (also known as MPH966, formerly known as AZD9668) is an orally administered, highly selective neutrophil elastase inhibitor, which differs significantly from cevelex in molecular structure, pharmacokinetic properties, and route of administration. Currently, Alvelestat is primarily used in clinical research for respiratory diseases, and there are no known technical solutions for its use in the treatment of cerebral hemorrhage.

[0007] In summary, there is an urgent need in this field to develop a treatment strategy that can effectively prevent secondary brain injury following cerebral hemorrhage. Summary of the Invention

[0008] This invention is the first to use time-stratified proteomics analysis of hematoma fluid / plasma in patients with cerebral hemorrhage to discover that NE is a key molecule in the progression of cerebral hemorrhage. Subsequently, the selective NE inhibitor Alvelestat was used for intervention, and animal experiments and transcriptomics were used to verify its effects in reducing cerebral edema, promoting hematoma absorption, reducing neuronal apoptosis, improving neurological functional outcomes, and regulating inflammatory and immune-related signaling pathways.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the invention provides the use of Alvelestat in the preparation of a medicament for the treatment of cerebral hemorrhage.

[0010] Alvelestat (also known as MPH966, formerly known as AZD9668) is an oral, highly selective inhibitor of neutrophil elastase with an IC50 of 12 nM and superior selectivity compared to other serine proteases.

[0011] Furthermore, the drug is used to treat secondary brain injury following cerebral hemorrhage.

[0012] Furthermore, the secondary brain injury is cerebral edema.

[0013] Furthermore, the secondary brain injury is neuronal apoptosis.

[0014] Furthermore, the secondary brain injury is a neurological deficit.

[0015] In the specific implementation plan, the cerebral edema refers to vasogenic edema and cytotoxic edema that occur in the area surrounding the hematoma after cerebral hemorrhage; the neuronal apoptosis refers to TUNEL positive staining of NeuN-positive neurons in the penumbra area surrounding the hematoma; the neurological deficit includes, but is not limited to, motor dysfunction, sensory dysfunction and cognitive dysfunction, which can be assessed by modified neurological deficit score, right turn count test and forelimb use asymmetry test.

[0016] Furthermore, the dosage of Alvelestat in the animal model of cerebral hemorrhage is from 1 mg / kg to 5 mg / kg. This dosage range was determined based on experiments using a mouse model of cerebral hemorrhage, where 1 mg / kg is the low-dose group and 5 mg / kg is the high-dose group. Experimental results showed that the 5 mg / kg dosage group had a more significant improvement effect. Those skilled in the art can extrapolate this dosage range to the human therapeutic dose according to the animal equivalent dose conversion rules.

[0017] A second aspect of the invention provides the use of Alvelestat in the preparation of a medicament for inhibiting inflammatory immune-related signaling pathways after cerebral hemorrhage, wherein the signaling pathway is selected from one or more of the TNF signaling pathway, JAK-STAT signaling pathway, NOD-like receptor signaling pathway, and chemokine signaling pathway.

[0018] In a specific implementation, the TNF signaling pathway includes a TNF-α-mediated inflammatory cascade; the JAK-STAT signaling pathway includes JAK2 / STAT3 phosphorylation activation; the NOD-like receptor signaling pathway includes NLRP3 inflammasome activation; and the chemokine signaling pathway includes upregulation of CXCL and CCL family chemokine expression. Alvelestat reduces inflammatory damage following cerebral hemorrhage by inhibiting the activity of neutrophil elastase, thereby inhibiting the activation of one or more of the above signaling pathways.

[0019] A third aspect of the invention provides a pharmaceutical composition for treating cerebral hemorrhage, the pharmaceutical composition comprising Alvelestat and a pharmaceutically acceptable carrier.

[0020] Furthermore, the drug is an injectable or oral formulation. Alvelestat has good oral bioavailability, and the oral formulation is more suitable for pre-hospital emergency care and long-term rehabilitation of patients with cerebral hemorrhage; the injectable formulation (such as intraperitoneal injection) can be administered rapidly during the acute phase of cerebral hemorrhage, and its efficacy was verified by intraperitoneal injection in this embodiment of the invention.

[0021] Furthermore, the pharmaceutical composition may also contain one or more other active ingredients selected from anti-inflammatory drugs, antioxidants, or neuroprotective agents to achieve synergistic therapeutic effects.

[0022] The beneficial effects of this invention are as follows: Compared to existing technologies, this invention first achieves a breakthrough in its technical approach. Instead of directly intervening based on known targets, it utilizes time-stratified proteomics analysis of hematoma fluid / plasma from patients with cerebral hemorrhage to systematically screen and validate neutrophil elastase (NE) as a key molecule in the progression of cerebral hemorrhage from clinical samples, establishing a complete technical chain of "target discovery in clinical samples—animal experimental validation—transcriptomics mechanism analysis." Based on this, this invention clarifies NE as a more specific and druggable intervention target, employing the highly selective NE inhibitor Alvelestat for precise intervention. Compared to the non-specific degradation strategies targeting the entire NETs or extracellular DNA backbone in existing technologies, this approach offers stronger targeting and potential safety. Animal experiments have demonstrated that Alvelestat can significantly reduce cerebral edema after cerebral hemorrhage, promote hematoma absorption, reduce neuronal apoptosis, and improve neurological outcomes. Transcriptomics analysis further reveals that it exerts its neuroprotective effect by inhibiting multiple inflammatory and immune-related signaling pathways, including TNF, JAK-STAT, NOD-like receptors, and chemokines, with a clear mechanism. Furthermore, Alvelestat is an oral formulation, which is more convenient for clinical application compared to existing NE inhibitors (such as cevelexat) that require intravenous administration. Currently, there is a lack of specific treatments for cerebral hemorrhage; this invention provides a novel treatment strategy for this serious disease, demonstrating significant innovation and translational application value. Attached Figure Description

[0023] Figure 1 This image shows the results of proteomics analysis of hematoma fluid / plasma in patients with cerebral hemorrhage. Figure 1 A is the principal component analysis diagram; Figure 1 B is a heatmap of differential protein hierarchical clustering; Figure 1 C represents the GO enrichment analysis plot; Figure 1 D is a KEGG pathway enrichment analysis diagram; Figure 1 E is a heatmap of protein expression related to neutrophil extracellular traps (NETs); Figure 1 F is a volcano diagram.

[0024] Figure 2 Figure showing the experimental results of Alvelestat improving brain injury and neurological function outcomes in mice with cerebral hemorrhage. (The figure is missing from the original text.) Figure 2 A is a schematic diagram of the animal experiment process; Figure 2 B shows representative magnetic resonance imaging images of mice in each group at baseline, day 3, and day 12; Figure 2 C shows the results of quantitative analysis of cerebral edema on day 3; Figure 2 Figure D shows the results of the hematoma volume reduction ratio analysis on day 12; Figure 2 E is a representative image of TUNEL / NeuN immunofluorescence staining of brain tissue in each group; Figure 2 F shows the results of quantitative analysis of TUNEL and NeuN double-positive cells; Figure 2 G represents the behavioral test results of the number of right turns; Figure 2 H represents the modified neurological deficit score results; Figure 2 Figure I shows the results of the forelimb asymmetry test.

[0025] Figure 3 This figure shows the experimental results of transcriptome sequencing analysis of the effects of Alvelestat intervention on gene expression in the brain tissue of mice with cerebral hemorrhage. Figure 3 A is the principal component analysis diagram; Figure 3 B is a heatmap of hierarchical clustering of differentially expressed genes; Figure 3 C represents the GO enrichment analysis plot; Figure 3 D is a KEGG pathway enrichment analysis diagram; Figure 3 E is a chord diagram of differentially expressed genes and the enriched KEGG pathway; Figure 3 F is the Venn diagram; Figure 3 G represents a ring-shaped heatmap; Figure 3 H represents a volcano diagram; Figure 3 I is a graph representing gene set enrichment analysis. Detailed Implementation

[0026] 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.

[0027] Example 1: Proteomic analysis of hematoma fluid / plasma in patients with cerebral hemorrhage This embodiment aims to clarify the changes in molecular characteristics of hematoma fluid / plasma at different time stages after cerebral hemorrhage by performing proteomics analysis on samples from patients with cerebral hemorrhage, and to screen key molecules related to disease progression.

[0028] Cerebral hematoma fluid samples were collected from 16 patients with cerebral hemorrhage, and their plasma was separated. Patients were divided into a short-term (Short) and a long-term (>30 h) group based on the time from onset to sampling. Proteomics sequencing was used to analyze the samples from both groups, and principal component analysis (PCA), differential protein clustering analysis, GO enrichment analysis, KEGG pathway enrichment analysis, and volcano plot analysis were performed. Differentially expressed proteins related to neutrophil extracellular traps were highlighted.

[0029] PCA results showed a significant separation in protein expression profiles between the shorter and longer time-to-onset groups, suggesting significant differences in molecular characteristics of hematoma fluid in patients with cerebral hemorrhage within different time windows. Figure 1 A). The differential protein clustering heatmap showed that the two groups of samples had different overall protein expression patterns. Figure 1 B). GO enrichment analysis showed that the differentially expressed proteins were mainly involved in biological processes, cellular components, and molecular functions such as protein complex assembly, organelle organization, extracellular vesicles, ribosome-associated complexes, and nucleotide binding. Figure 1 C). KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in multiple inflammation and immune-related pathways, with the neutrophil extracellular trap formation pathway being significantly enriched (C). Figure 1 D). The heatmap of NETs-related proteins showed that multiple NETs-related proteins generally showed an upregulation trend in the group with longer disease duration. Figure 1 E). Volcano plot analysis showed that, compared with the group with shorter onset time, the group with longer onset time had extensive differential protein expression, with 1059 proteins significantly upregulated and 857 proteins significantly downregulated. Among them, ELANE was one of the differentially expressed proteins upregulated in the group with longer onset time. Figure 1 F).

[0030] The above results indicate that as the time since the onset of cerebral hemorrhage increases, the expression of NETs-related molecules in the hematoma fluid of patients increases, among which neutrophil elastase (NE) may be involved in the pathological process after cerebral hemorrhage.

[0031] Example 2: Effects of Alvelestat on Brain Injury and Neurological Function in Mice with Cerebral Hemorrhage A mouse model of intracranial hemorrhage was established. Mice were anesthetized with isoflurane (1.5-2% maintenance) and placed in a stereotactic frame. Under aseptic conditions, an incision was made along the midline of the scalp, and a hole was drilled 3 mm lateral to the skull (right) and 0.2 mm anterior to the anterior fontanelle. A 26-gauge needle (Hamilton syringe; Hamilton, Reno, NV, USA) was placed 5.5 mm below the surface of the skull, and 25 μL of autologous venous blood (obtained from the mouse's medial canthal vein) was injected into the striatum. The needle was left in place for 5 minutes after injection. A metal screw (model MX-080-2; Small Parts, Miami Lakes, FL, USA) was inserted to the thickness of the skull. The scalp wound was treated with esophagine and sutured. To minimize the invasiveness and duration of anesthesia, no intubation was performed on the mice during the procedure. Throughout the procedure (approximately 30 minutes), a heating blanket was used to maintain the core temperature at around 37.0°C. After modeling, mice were randomly divided into an ICH group, a low-dose Alvelestat group (1 mg / kg), and a high-dose Alvelestat group (5 mg / kg). One hour after modeling, mice were administered Alvelestat via intraperitoneal injection at doses of 1 mg / kg and 5 mg / kg, respectively. Baseline behavioral assessments were performed one day prior to modeling, and behavioral evaluations were conducted on days 1, 3, 7, and 12 post-modeling. Magnetic resonance imaging (MRI) was performed on days 1, 3, and 7 to assess changes in cerebral edema and hematoma. TUNEL / NeuN immunofluorescence staining was used to assess neuronal apoptosis around the hematoma on day 3. Behavioral assessments included a modified neurological deficit score, a right turn count test, and a forelimb asymmetry use test. Figure 2 A).

[0032] MRI results showed that, compared with the ICH group, the extent of brain injury was reduced after Alvelestat treatment ( Figure 2 B). Quantitative analysis showed that Alvelestat treatment reduced cerebral edema levels on day 3 (B). Figure 2 C), and promotes hematoma volume reduction on day 12 ( Figure 2 D). Histological results showed that Alvelestat treatment reduced the number of TUNEL-positive cells around the hematoma and decreased the proportion of TUNEL and NeuN double-positive cells. Figure 2 E, Figure 2 F). Behavioral results showed that, compared with the cerebral hemorrhage model group, the Alvelestat treatment group had fewer right turns (F). Figure 2 G), improved neurological deficit score reduction ( Figure 2 H), Improvement of forelimb asymmetry ( Figure 2 I). The above-mentioned improvement was more pronounced in the 5 mg / kg dose group than in the 1 mg / kg dose group.

[0033] The above results indicate that Alvelestat can reduce cerebral edema and neuronal apoptosis after cerebral hemorrhage, promote hematoma absorption, and improve neurological functional outcomes in mice.

[0034] Example 3: Effects of Alvelestat intervention on the transcriptome of brain tissue in mice with cerebral hemorrhage RNA was extracted from brain tissues of mice in the control group (Con), the intracerebral hemorrhage model group (ICH), and the Alvelestat treatment group (AT), and transcriptome sequencing was performed. Principal component analysis (PCA) and differentially expressed gene clustering heatmaps were used to assess the overall transcriptional differences among the groups. GO and KEGG enrichment analyses were performed on differentially expressed genes between the Alvelestat treatment group and the ICH model group, and string plots were used to illustrate the correspondence between differentially expressed genes and enriched pathways. Venn plots were used to analyze the intersection genes between the ICH model group and the control group, and between the Alvelestat treatment group and the ICH model group, and ring heatmaps were used to illustrate the expression patterns of these intersection genes in each group. Volcano plots and gene set enrichment analysis were used to assess the impact of Alvelestat intervention on inflammatory and immune-related signaling pathways.

[0035] PCA results showed significant separation at the transcriptional level among the control group, the cerebral hemorrhage model group, and the Alvelestat treatment group. Figure 3 A). The differentially expressed gene clustering heatmap showed that there were significantly different gene expression patterns between the cerebral hemorrhage model group and the control group, and some abnormally expressed genes showed a reversion trend after Alvelestat treatment. Figure 3 B). GO enrichment analysis showed that differentially expressed genes after Alvelestat intervention were mainly involved in immune inflammation-related functions such as positive regulation of immune responses, leukocyte migration, cytokine receptor binding, chemokine activity, and phagocytosis-related cell structures. Figure 3 C). KEGG pathway analysis showed that differentially expressed genes were significantly enriched in inflammatory and immune-related pathways such as cytokine-cytokine receptor interactions, chemokine signaling pathways, TNF signaling pathways, JAK-STAT signaling pathways, NOD-like receptor signaling pathways, and phagosomes. Figure 3 D). The string diagram further suggests that multiple differentially expressed genes are involved in the regulation of the above pathways ( Figure 3 E). The Venn diagram showed 115 overlapping genes between the cerebral hemorrhage model group and the control group, and between the Alvelestat treatment group and the cerebral hemorrhage model group. Figure 3 F), the circular heatmap further shows that these overlapping genes partially exhibit a regression trend after Alvelestat treatment (F). Figure 3G). The volcano plot showed that among the differentially expressed genes between the Alvelestat treatment group and the cerebral hemorrhage model group, 16 genes were upregulated and 110 genes were downregulated, among which Mmp9 was one of the significantly differentially expressed genes. Figure 3 H). Gene set enrichment analysis showed that, compared with the cerebral hemorrhage model group, Alvelestat intervention suppressed multiple inflammation and immune-related pathways, including the TNF signaling pathway, JAK-STAT signaling pathway, NOD-like receptor signaling pathway, phagosomes, and cytokine-receptor interactions. Figure 3 I).

[0036] The above results indicate that Alvelestat intervention can significantly regulate the inflammatory immune-related transcriptional program following cerebral hemorrhage and partially reverse the abnormal gene expression changes induced by cerebral hemorrhage. Although embodiments of the present invention have been shown and described above, it is understood that these 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. Application of Alvelestat in the preparation of drugs for the treatment of cerebral hemorrhage.

2. The application according to claim 1, characterized in that, The drug is used to treat secondary brain injury following cerebral hemorrhage.

3. The application according to claim 2, characterized in that, The secondary brain injury is cerebral edema.

4. The application according to claim 2, characterized in that, The secondary brain injury is neuronal apoptosis.

5. The application according to claim 2, characterized in that, The secondary brain injury is a neurological deficit.

6. The application according to claim 1, characterized in that, The dosage of Alvelestat in the animal model of cerebral hemorrhage is 1 mg / kg to 5 mg / kg.

7. The use of Alvelestat in the preparation of drugs for inhibiting inflammatory immune-related signaling pathways after cerebral hemorrhage, wherein the signaling pathway is selected from one or more of the TNF signaling pathway, JAK-STAT signaling pathway, NOD-like receptor signaling pathway, and chemokine signaling pathway.

8. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition comprising Alvelestat and a pharmaceutically acceptable carrier.

9. The application according to claim 1, characterized in that, The drug is an injectable or oral formulation.

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