Use of dipeptidyl peptidase 1 inhibitors for the preparation of a medicament for the treatment of cerebral hemorrhage

Abstract

The application discloses application of a dipeptidyl peptidase 1 inhibitor in preparation of a medicine for treating cerebral hemorrhage. The application establishes dipeptidyl peptidase 1 as an intervention target for cerebral hemorrhage treatment based on proteomic analysis of hematoma liquid of a cerebral hemorrhage patient. The dipeptidyl peptidase 1 inhibitor Brensocatib can improve neurological impairment after cerebral hemorrhage, reduce cerebral edema, promote hematoma absorption, reduce neuron damage, and inhibit inflammatory cell infiltration. The application provides a new drug intervention strategy for cerebral hemorrhage treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of dipeptidyl peptidase-1 inhibitors in the preparation of drugs for treating cerebral hemorrhage. Background Technology

[0002] Intracerebral hemorrhage (ICH) is one of the most deadly and disabling types of stroke. Currently, clinical treatment mainly focuses on comprehensive supportive care, including blood pressure management, intracranial pressure control, and surgical hematoma evacuation. These measures are primarily used to limit hematoma expansion, alleviate mass effect, and manage serious complications, but they are not yet able to directly prevent the continuous secondary brain injury that occurs after cerebral hemorrhage.

[0003] Following a brain hemorrhage, neutrophils are among the first peripheral immune cells to enter the hematoma and surrounding brain tissue. Activated neutrophils can participate in blood-brain barrier disruption, cerebral edema, and neuronal damage by degranulating to release various serine proteases and forming neutrophil extracellular traps (NETs).

[0004] Neutrophil serine proteases (NSPs) mainly include neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG), which are stored in azurophilic granules of neutrophils. All three proteases depend on dipeptidyl peptidase 1 (DPP1, also known as cathepsin C) to cleave the N-terminal dipeptide of their precursors during neutrophil bone marrow development and maturation, in order to be converted into catalytically active mature proteases. Therefore, dipeptidyl peptidase 1 is located upstream of the common activation process of these three major neutrophil serine proteases and is a key molecule regulating the proteolytic activity of mature neutrophils.

[0005] Brensocatib is an oral, selective, and reversible dipeptidyl peptidase-1 inhibitor that has been clinically studied in respiratory diseases such as bronchiectasis, reducing the activity of various neutrophil serine proteases in patients. However, these studies all focus on chronic respiratory inflammation, whose pathological processes differ fundamentally from those following cerebral hemorrhage, including the hematoma microenvironment, blood-brain barrier disruption, and neuronal damage. In the field of neurological diseases, although studies have explored the role of dipeptidyl peptidase-1 inhibitors in cerebral ischemia models, the pathological processes involved in cerebral hemorrhage, such as hematoma formation, erythrocyte lysis, and hemoglobin toxicity, are significantly different from those in ischemic stroke.

[0006] To date, there have been no reports of using dipeptidyl peptidase-1 inhibitors for the treatment of cerebral hemorrhage. Summary of the Invention

[0007] The technical problem to be solved by the present invention is the lack of specific drugs in the prior art that can effectively improve the long-term prognosis of patients with cerebral hemorrhage, and the fact that dipeptidyl peptidase 1 inhibitors are still a blank in the field of cerebral hemorrhage treatment. The present invention provides the application of dipeptidyl peptidase 1 inhibitors in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Application of dipeptidyl peptidase-1 inhibitors in the preparation of drugs for the treatment and / or relief of cerebral hemorrhage.

[0009] In some embodiments of the present invention, the dipeptidyl peptidase 1 inhibitor is brensocatib or a pharmaceutically acceptable salt thereof. Brensocatib (also known as INS1007, AZD7986) is an oral, selective, reversible dipeptidyl peptidase 1 inhibitor. The molecular formula of this compound is C1. 23 H 24 N4O4, with a molecular weight of 420.46 and CAS number 1802148-05-5. Pharmaceutically acceptable salts of Brensocatib include inorganic or organic acid salts such as hydrochloride, sulfate, phosphate, methanesulfonate, maleate, tartrate, and citrate. The chemical structure of Brensocatib (Brensocatib) is shown below. Figure 5 It can be used in its original compound form, or in the form of its pharmaceutically acceptable salt, solvate, hydrate, crystal form, or prodrug, all of which can be used to prepare drugs for treating and / or alleviating cerebral hemorrhage.

[0010] In this invention, dipeptidyl peptidase 1 is a common upstream enzyme for the maturation of three neutrophil serine proteases: elastase, protease 3, and cathepsin G. Inhibiting dipeptidyl peptidase 1 activity can simultaneously reduce the activation levels of these three serine proteases. Based on this common mechanism of action, those skilled in the art can reasonably expect that not only Brensocatib, a specific dipeptidyl peptidase 1 inhibitor, has a therapeutic effect on cerebral hemorrhage, but rather that compounds with dipeptidyl peptidase 1 inhibitory activity may produce similar therapeutic effects on cerebral hemorrhage through the same upstream regulatory mechanism. Therefore, the technical solutions claimed in this invention cover various compounds with dipeptidyl peptidase 1 inhibitory activity and their pharmaceutically acceptable forms.

[0011] In some embodiments of the present invention, the drug is used to alleviate secondary brain injury following cerebral hemorrhage. After cerebral hemorrhage, the brain tissue surrounding the hematoma may develop one or more pathological changes, including cerebral edema, impaired hematoma absorption, blood-brain barrier disruption, neuronal degeneration or death, inflammatory cell infiltration, neutrophil extracellular trap-related damage, microglia / macrophage activation, or neurological deficits. Dipeptidyl peptidase-1 inhibitors can alleviate the aforementioned secondary brain injury.

[0012] In some embodiments of the present invention, the drug is used to achieve at least one of the following effects: reducing cerebral edema, promoting hematoma absorption, reducing neuronal damage, improving neurological deficits, or reducing inflammatory cell infiltration in brain tissue. Specifically, reducing cerebral edema includes reducing cerebral edema around the hematoma; promoting hematoma absorption includes accelerating the reduction of hematoma volume within the brain; reducing neuronal damage includes reducing neuronal degeneration and / or death around the hematoma; improving neurological deficits includes improving motor function, sensory function, balance function, and / or reflex function after cerebral hemorrhage; and reducing inflammatory cell infiltration in brain tissue includes reducing the infiltration of inflammatory cells such as neutrophils, monocytes / macrophages around the hematoma.

[0013] In some embodiments of the present invention, the cerebral hemorrhage includes one of the following: spontaneous cerebral hemorrhage, hypertensive cerebral hemorrhage, basal ganglia hemorrhage, lobar hemorrhage, cerebellar hemorrhage, brainstem hemorrhage, or deep cerebral hemorrhage. In some embodiments of the present invention, the cerebral hemorrhage is spontaneous. In some embodiments of the present invention, the cerebral hemorrhage is hypertensive cerebral hemorrhage.

[0014] In some embodiments of the invention, the drug is administered within 72 hours of the onset of cerebral hemorrhage. In some embodiments of the invention, the drug is administered within 48 hours of the onset of cerebral hemorrhage. In some embodiments of the invention, the drug is administered within 24 hours of the onset of cerebral hemorrhage. In some embodiments of the invention, the drug is administered within 6 hours of the onset of cerebral hemorrhage.

[0015] In some embodiments of the present invention, the drug is administered once daily. In some embodiments of the present invention, the drug is administered continuously for 5 to 14 days. In some embodiments of the present invention, the drug is administered continuously for 7 days.

[0016] In some embodiments of the present invention, the drug is an oral formulation. The oral formulation includes one of tablets, capsules, granules, suspensions, or oral solutions. In some embodiments of the present invention, the drug is an injectable formulation. In some embodiments of the present invention, the drug is a sustained-release formulation, a controlled-release formulation, or a nanoformulation.

[0017] In some embodiments of the invention, the medicament further comprises one or more pharmaceutically acceptable excipients. The excipients include one or more of fillers, binders, disintegrants, lubricants, coating materials, or release modifiers.

[0018] Further, when the subject of the present invention is a human, the dosage of the dipeptidyl peptidase 1 inhibitor is 0.01 to 2.00 mg / kg body weight; preferably, the dosage is 0.01 to 1.50 mg / kg body weight; more preferably, the dosage is 0.01 to 1.00 mg / kg body weight; more preferably, the dosage is 0.02 to 1.00 mg / kg body weight.

[0019] In some embodiments of the present invention, the dosage of the dipeptidyl peptidase 1 inhibitor is 1 to 25 mg / kg body weight.

[0020] In some embodiments of the present invention, the dose of the dipeptidyl peptidase-1 inhibitor is 5 mg / kg body weight. Based on body surface area conversion (animal dose (mg / kg) × Km (animal) / Km (human), where mouse Km=3 and adult human Km=37), a dose of 5 mg / kg in mice is approximately equivalent to 0.4 mg / kg in humans. Those skilled in the art can determine the equivalent dose range suitable for humans based on animal experimental data using conventional pharmacokinetic and dose conversion methods.

[0021] Beneficial effects This invention marks the first discovery that dipeptidyl peptidase-1 inhibitors can be used to prepare drugs for treating and / or alleviating cerebral hemorrhage, filling a gap in this technological field. Based on time-stratified proteomics analysis of hematoma fluid from cerebral hemorrhage patients, this invention is the first to discover a synchronous increase in three dipeptidyl peptidase-1-dependent neutrophil serine proteases during the course of cerebral hemorrhage, thus establishing dipeptidyl peptidase-1 as an intervention target for the treatment of cerebral hemorrhage. This invention, through an animal model of cerebral hemorrhage, demonstrates that dipeptidyl peptidase-1 inhibitors can improve neurological deficits after cerebral hemorrhage, reduce cerebral edema, promote hematoma absorption, reduce neuronal damage, and inhibit inflammatory cell infiltration. Simultaneously, transcriptomic results show that they can inhibit inflammatory immune pathways such as neutrophil extracellular trap formation, complement coagulation, cell adhesion, and Toll-like receptor signaling, validating their mechanism of action at the molecular level. Compared with existing intervention strategies that target only a single terminal effector molecule (such as neutrophil elastase), this invention targets dipeptidyl peptidase 1, which can simultaneously regulate the activity of multiple neutrophil serine proteases, with a wider range of effects and more systematic regulation, providing a new intervention strategy for cerebral hemorrhage, a disease for which there are currently no specific therapeutic drugs. Attached Figure Description

[0022] Figure 1The results of proteomic analysis of hematoma fluid from a patient with cerebral hemorrhage in Example 1 of this invention. Among them: Figure 1 A is the principal component analysis (PCA) plot; Figure 1 B is a hierarchical clustering heatmap of differentially expressed proteins; Figure 1 C represents the volcano plot of differentially expressed proteins; Figure 1 D represents the GO enrichment analysis result; Figure 1 E represents the results of KEGG pathway enrichment analysis; Figure 1 F is the GSEA enrichment curve; Figure 1 G is a chord diagram of differentially expressed proteins and the KEGG enrichment pathway; Figure 1 H represents the intergroup quantitative comparison results of ELANE protein expression levels; Figure 1 I represents the intergroup quantitative comparison results of PRTN3 protein expression levels; Figure 1 J represents the intergroup quantitative comparison results of CTSG protein expression levels.

[0023] Figure 2 This presents the results of the dose and administration time screening of the DPP1 inhibitor Brensocatib in mice with cerebral hemorrhage in Example 2 of this invention. Wherein: Figure 2 A is the flowchart of the dose screening experiment; Figure 2 B represents the effect of different doses of BC on the mNSS score of mice with cerebral hemorrhage; Figure 2 C represents the effect of different doses of BC on the proportion of right turns in mice with cerebral hemorrhage; Figure 2 D represents the effect of different doses of BC on asymmetric forelimb use in mice with cerebral hemorrhage; Figure 2 E is the flowchart for the drug administration timeline screening experiment; Figure 2 F represents the effect of different drug administration durations on the mNSS score of mice with cerebral hemorrhage; Figure 2 G represents the effect of different drug administration durations on the proportion of right turns in mice with cerebral hemorrhage; Figure 2 H represents the effect of different drug administration times on the asymmetry of forelimb use in mice with cerebral hemorrhage.

[0024] Figure 3The results of the effects of the DPP1 inhibitor Brensocatib on brain injury and inflammatory cell infiltration in mice with cerebral hemorrhage in Example 3 of this invention are shown. Wherein: Figure 3 A is a flowchart of animal experiments; Figure 3 B shows the T2-weighted magnetic resonance imaging images of mice in each group; Figure 3 C represents the quantitative analysis results of the proportion of cerebral edema; Figure 3 D represents the quantitative analysis result of the hematoma volume reduction ratio; Figure 3 E represents the mNSS score of mice in each group; Figure 3 F represents the proportion of mice turning right in each group; Figure 3 G represents the asymmetric results of forelimb use in each group of mice; Figure 3 H is a Fluoro-Jade C staining image of brain tissue surrounding hematoma in mice of each group (scale bar = 50 μm). Figure 3 I represents the quantitative analysis results of the number of Fluoro-Jade C positive cells; Figure 3 J shows the MPO / NE immunofluorescence staining images of brain tissue surrounding hematomas in mice from each group (left low-power image scale bar = 200 μm, right magnified image scale bar = 50 μm). Figure 3 K represents the quantitative analysis result of the number of MPO-positive cells; Figure 3 L represents the quantitative analysis result of the number of NE-positive cells; Figure 3 M represents the quantitative analysis result of the number of MPO / NE double-positive cells; Figure 3 N is an immunohistochemical staining image of IBA-1 in the brain tissue surrounding the hematoma of mice in each group (low magnification on the left, scale bar = 200 μm; magnification on the right, scale bar = 50 μm). Figure 3 O represents the quantitative analysis result of the number of IBA-1 positive cells.

[0025] Figure 4 The results of the effect of the DPP1 inhibitor Brensocatib on the transcriptome of brain tissue in mice with cerebral hemorrhage in Example 4 of this invention are shown. Wherein: Figure 4 A is the principal component analysis (PCA) plot; Figure 4 B is a volcano diagram of differentially expressed genes; Figure 4 C represents the hierarchical clustering heatmap of differentially expressed genes; Figure 4 D represents the results of KEGG pathway enrichment analysis; Figure 4 E represents the GO enrichment analysis result; Figure 4 F is a chord diagram of differentially expressed genes and the KEGG enrichment pathway; Figure 4 G represents the GSEA enrichment analysis result; Figure 4 H represents the Venn diagram of the intersection genes; Figure 4 I represents the circular heatmap of intersecting gene expression; Figure 5 The chemical structure of Brensocatib is given. Detailed Implementation

[0026] The present invention will be described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in this invention are conventional methods; the biological materials, reagents, and instruments described are all commercially available unless otherwise specified.

[0027] The main materials and reagents used in this invention are sourced from the following sources: Brensocatib (hereinafter referred to as BC), as the drug intervention reagent in this experiment, was purchased from MedChemExpress; the experimental animals were SPF-grade male C57BL / 6J mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; anti-myelin basic protein (MBP, 1:400) antibody was purchased from Abcam (ab218011); anti-neutrophil elastase (NE, 1:200) antibody was purchased from Proteintech (27642-1-AP); anti-myeloperoxidase (MPO, 1:200) antibody was purchased from Servicebio (GB11224-100); anti-Iba-1 (1:200) antibody was purchased from Servicebio (GB12105-100).

[0028] Example 1: Dynamic proteomic analysis of hematoma fluid in patients with cerebral hemorrhage This embodiment aims to determine the changes in protein expression in the local inflammatory microenvironment of the hematoma at different time stages after cerebral hemorrhage by performing proteomics analysis on hematoma fluid samples from patients with cerebral hemorrhage at different onset times, and to screen inflammatory pathways and potential drug targets related to the progression of cerebral hemorrhage.

[0029] Hematoma fluid samples were collected from 22 patients with cerebral hemorrhage. Based on the time from the onset of cerebral hemorrhage to the collection of hematoma fluid, the samples were divided into a short onset group (Short group, n=11) and a long onset group (Long group, n=11) with 36 hours after the onset of the hemorrhage as the grouping cutoff.

[0030] Hematoma fluid samples collected from patients with cerebral hemorrhage underwent preprocessing and protein extraction, followed by quantitative proteomics analysis. Based on the quantitative results of proteins in each sample, the following analyses were performed sequentially: Principal component analysis (PCA) was used to compare the overall protein expression characteristics between the groups with shorter and longer onset times; differential protein clustering heatmaps were used to display the protein expression patterns of the two groups; volcano plots were used to analyze upregulated and downregulated differentially expressed proteins in the group with longer onset times compared to the group with shorter onset times; Gene Ontology (GO) enrichment analysis was performed on differentially expressed proteins, including biological process (BP), cellular component (CC), and molecular function (MF); Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on differentially expressed proteins; and gene set enrichment analysis was conducted. The GSEA analysis was used to assess the overall enrichment direction of related pathways in the group with a longer disease duration; a correspondence map between differentially expressed proteins and major enriched pathways was constructed to analyze key proteins involved in NET formation, phagocytosis and other inflammatory immune pathways; and expression heatmaps and intergroup quantitative comparisons were performed on three major downstream neutrophil serine proteases of DPP1, namely NE encoded by ELANE, PR3 encoded by PRTN3 and CatG encoded by CTSG.

[0031] PCA analysis showed that the shorter duration of illness was significantly different from the longer duration of illness in terms of overall protein expression profiles. Figure 1 A). Further differential protein clustering heatmaps revealed different protein expression patterns between the two groups. The group with longer disease duration exhibited a group of proteins with significantly upregulated expression, while other proteins showed decreased expression. Figure 1 B). Volcano plot results showed a large number of differentially expressed proteins between the longer and shorter disease duration groups, with some proteins significantly upregulated and others significantly downregulated in the longer disease duration group. Figure 1 C).

[0032] GO enrichment analysis showed that, in terms of biological processes, the differentially expressed proteins were mainly involved in processes such as nucleosome assembly, nucleosome organization, protein-DNA complex assembly, and protein complex remodeling; in terms of cellular components, the differentially expressed proteins were significantly enriched in cellular structures such as secretory granules, secretory granule lumen, cytoplasmic vesicle lumen, vesicle lumen, and nucleosomes; in terms of molecular function, the differentially expressed proteins were mainly involved in peptidase regulatory activity, peptidase inhibitory activity, endopeptidase regulatory activity, endopeptidase inhibitory activity, receptor ligand activity, and glycosaminoglycan binding. Figure 1 D).

[0033] KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in neutrophil extracellular trap formation, phagocytosis, complement and coagulation cascades, proteasomes, systemic lupus erythematosus, and other inflammatory immune-related pathways. Figure 1 E); among them, the neutrophil extracellular trap formation pathway has a high enrichment level and a large number of differentially expressed proteins, and is one of the most prominent inflammation-related pathways in the group with a longer disease duration.

[0034] GSEA results showed that the neutrophil extracellular trap formation pathway was significantly positively enriched in the group with a longer disease duration, with a normalized enrichment score (NES) of 2.87 and a p-value of 5.2 × 10⁻⁶. -17 ( Figure 1 F). Furthermore, the phagocytic pathway also showed significant positive enrichment, with an NES of 1.82 and a P-value of 4.0 × 10⁻⁶. -5 The systemic lupus erythematosus-related pathway and the Epstein-Barr virus infection-related pathway also showed positive enrichment. These pathways all contain a large number of histones, granulocytes, and inflammatory immune-related molecules, and their enrichment may reflect neutrophil degranulation, NET formation, cellular debris clearance, and a persistently enhanced immune response within the hematoma environment of cerebral hemorrhage.

[0035] String diagram analysis of differentially expressed proteins and the KEGG pathway revealed that multiple neutrophil granule proteins, oxidative stress-related proteins, and histones are involved in the NETs formation pathway, including ELANE, PRTN3, CTSG, MPO, MMP8, MMP9, NCF1, NCF2, NCF4, CYBB, PADI4, and various histone-related proteins. Figure 1 G).

[0036] Further analysis of neutrophil serine proteases showed that ELANE, PRTN3, and CTSG all exhibited a consistent upward trend in the group with longer disease duration. Protein expression heatmaps showed that the overall expression levels of ELANE, PRTN3, and CTSG were lower in the group with shorter disease duration, while all three proteins were elevated in the group with longer disease duration. Quantitative comparisons further showed that: neutrophil elastase NE encoded by ELANE was significantly elevated in the group with longer disease duration; protease 3 PR3 encoded by PRTN3 was significantly elevated in the group with longer disease duration; and cathepsin G CatG encoded by CTSG was significantly elevated in the group with longer disease duration. Figure 1 H, 1I, 1J).

[0037] The above results indicate that prolonged onset of cerebral hemorrhage is accompanied by significant activation of the NETs formation pathway in the hematoma microenvironment, and simultaneous increases in three major neutrophil serine proteases downstream of DPP1.

[0038] Example 2: Screening of the dosage and administration time of the DPP1 inhibitor Brensocatib in mice with cerebral hemorrhage. This embodiment aims to clarify the dosage and administration time for subsequent animal efficacy and mechanism studies by comparing the recovery of neurological function in mice under different doses and administration durations.

[0039] A basal ganglia hemorrhage model was established in C57BL / 6J mice using autologous blood injection. Specifically, after anesthetizing mice with isoflurane, they were fixed in a stereotaxic apparatus, and 25 μL of autologous venous blood from the inner canthus was slowly injected into the right basal ganglia region (coordinates: 0.2 mm posterior to the anterior fontanelle, 2.0 mm to the right, and 3.5 mm subdurally) to establish the hemorrhage model. The sham-operated group underwent only acupuncture without blood injection.

[0040] Mice with successfully induced cerebral hemorrhage were randomly divided into a cerebral hemorrhage model group (ICH group) and different Brensocatib (hereinafter referred to as BC) treatment groups (including three dose groups: 1 mg / kg, 5 mg / kg, and 10 mg / kg). Brensocatib was added to DMSO to prepare a stock solution, and PEG300, Tween 80, and 45% Saline were added as needed to prepare different working solutions. Administration was via intraperitoneal injection (10 mL / kg body weight). The first dose was administered 6 hours after modeling, followed by once daily for 7 consecutive days. Baseline behavioral assessments were performed before modeling, and behavioral assessments were performed on days 1, 3, 7, and 14 after modeling. Assessment indicators included modified neurological deficit score (mNSS score), right turn rate, and forelimb asymmetry.

[0041] Behavioral results showed no significant differences in mNSS scores, right turn rate, and forelimb asymmetry among the groups before modeling, indicating consistent baseline neurological function across groups. After cerebral hemorrhage modeling, the ICH group showed significantly increased mNSS scores, increased right turn rate, and aggravated forelimb asymmetry, indicating significant unilateral motor dysfunction and neurological deficits caused by cerebral hemorrhage. On day 1 post-modeling, there were no significant differences in behavioral indicators between the BC treatment groups and the ICH group. By day 3 post-modeling, the 5 mg / kg and 10 mg / kg treatment groups began to show improvement in some behavioral indicators. By days 7 and 14 post-modeling, the neurological function improvement was more pronounced in the 5 mg / kg and 10 mg / kg treatment groups, manifested as decreased mNSS scores, decreased right turn rate, and reduced forelimb asymmetry. Figure 2 A-2D).

[0042] Since the improvement effect of the 1 mg / kg group was relatively limited, and the 10 mg / kg group did not show a stable and clear additional benefit compared with the 5 mg / kg group, after taking into account the need to reduce the dosage, reduce the potential drug burden, and improve the feasibility of subsequent experimental protocols, 5 mg / kg was finally selected as the dosage for subsequent experiments.

[0043] A candidate dose of 5 mg / kg was selected to further compare the effects of continuous administration for 7 days and 14 days on the recovery of neurological function after intracerebral hemorrhage. The experiment included an ICH group, a 7-day continuous BC administration group, and a 14-day continuous BC administration group. Baseline behavioral assessments were performed before modeling, and behavioral assessments were conducted on days 1, 3, 7, and 14 after modeling. The assessment indicators included modified neurological deficit score (mNSS score), the proportion of right turns, and forelimb asymmetry.

[0044] In the drug administration duration screening experiment, there were no significant differences in behavioral indicators among the groups of mice before modeling. After cerebral hemorrhage, the mNSS score, the proportion of right turns, and the asymmetry in forelimb use were significantly increased in the ICH group, indicating that the model was successfully established. Compared with the ICH group, both 7-day and 14-day continuous administration of BC improved neurological deficits after cerebral hemorrhage. Both drug administration durations reduced the mNSS score, decreased the proportion of right turns, and improved the asymmetry in forelimb use. Figure 2 (E-2H). However, compared with 7 days of administration, extending the administration to 14 days did not produce a stable and clear additional improvement advantage in the main behavioral indicators. Therefore, the present invention selected 7 consecutive days of administration as the administration period for subsequent experiments.

[0045] Example 3: Effects of the DPP1 inhibitor Brensocatib on brain injury and inflammatory cell infiltration in mice with cerebral hemorrhage. This embodiment aims to further verify the effects of Brensocatib on cerebral edema, hematoma absorption, neurological deficits, neuronal damage, and inflammatory cell infiltration after cerebral hemorrhage.

[0046] After establishing a mouse model of cerebral hemorrhage, mice were divided into a cerebral hemorrhage model group (ICH group) and a BC treatment group (ICH+BC group). The BC treatment group was treated with 5 mg / kg Brensocatib for 7 consecutive days. Baseline behavioral assessments were performed before modeling, and behavioral evaluations were conducted on days 1, 3, 7, and 14 after modeling, including modified neurological deficit score (mNSS), forelimb asymmetry test, and right turn count test. Magnetic resonance imaging (MRI) was performed on days 3 and 14 after modeling to assess cerebral edema and hematoma absorption, respectively. In addition, brain tissue was harvested for histological examination. Fluoro-Jade C staining was used to evaluate neuronal degeneration; MPO / NE immunofluorescence was used to detect neutrophils and neutrophil serine protease-related positive cells; and IBA-1 immunohistochemical staining was used to evaluate microglia / macrophage activation and infiltration.

[0047] Behavioral results showed that all mice had increased mNSS scores, increased forelimb asymmetry, and increased right turns before drug administration after modeling. Compared with the ICH group, the BC treatment group showed decreased mNSS scores, reduced right turns, and improved forelimb asymmetry at multiple observation time points, indicating that BC can promote the recovery of neurological function after cerebral hemorrhage. Figure 3 EG).

[0048] MRI results showed that the extent of brain hemorrhage was reduced in the BC group compared to the ICH group. Quantitative results on day 3 showed that BC treatment significantly reduced the proportion of cerebral edema; analysis of hematoma volume changes on day 14 showed that the proportion of hematoma volume reduction was higher in the BC treatment group than in the ICH group. Figure 3 BD).

[0049] Fluoro-Jade C staining results showed a large number of Fluoro-Jade C-positive cells in the brain tissue surrounding the hematoma in the ICH group. Compared with the ICH group, the number of Fluoro-Jade C-positive cells was significantly reduced in the BC-treated group. Figure 3 HI).

[0050] Immunofluorescence results showed that the ICH group had a large number of MPO-positive and NE-positive cells in the brain tissue surrounding the hematoma, and also a significant number of MPO / NE double-positive cells. Compared with the ICH group, the BC-treated group showed a significant reduction in the number of MPO-positive cells, NE-positive cells, and MPO / NE double-positive cells. Figure 3 JM).

[0051] IBA-1 immunohistochemical staining showed a significant increase in IBA-1 positive cells in the brain tissue surrounding the hematoma in the ICH group, while the number of IBA-1 positive cells was significantly reduced in the BC treatment group. Figure 3 NO).

[0052] The above results indicate that the DPP1 inhibitor BC can reduce cerebral edema after cerebral hemorrhage, promote hematoma absorption, improve neurological deficits, reduce neuronal degeneration and damage, and inhibit neutrophil-related inflammatory responses and microglia / macrophage activation.

[0053] Example 4: Effects of the DPP1 inhibitor Brensocatib on the transcriptome of brain tissue in mice with cerebral hemorrhage This embodiment aims to clarify the effect of the DPP1 inhibitor Brensocatib on the molecular transcriptional characteristics of brain tissue after cerebral hemorrhage, and to explore at the molecular transcriptional level whether Brensocatib intervention can improve secondary inflammatory brain injury after cerebral hemorrhage.

[0054] After establishing a mouse model of intracerebral hemorrhage, three groups were established: the Sham group (sham-operated group), the ICH group, and the BC treatment group (5 mg / kg, administered for 7 consecutive days). Reference data (Exp Neurol. 2026 Apr;398:115650.) showed a rapid transcriptional response after intracerebral hemorrhage, with the inflammatory response typically peaking on day 3 post-hemorrhage. Therefore, on day 3 after administration, RNA was extracted from brain tissue surrounding the hematoma and subjected to transcriptome sequencing. Subsequently, principal component analysis (PCA), differentially expressed gene volcano plots, differential gene clustering heatmaps, GO enrichment analysis, KEGG pathway enrichment analysis, differential gene-pathway chord maps, GSEA analysis, Venn intersection gene analysis, and intersection gene expression heatmap analysis were performed to evaluate the regulatory effect of brensocatib on ICH-induced transcriptional abnormalities.

[0055] PCA results showed that the Sham, ICH, and BC groups were significantly separated at the transcriptional level. Figure 4 A) indicates that cerebral hemorrhage can induce significant changes in the transcriptome of brain tissue, and the sample distribution after BC intervention is different from that of the ICH group, suggesting that BC can alter the overall gene expression characteristics after cerebral hemorrhage.

[0056] Differential gene clustering heatmaps showed that the ICH group had a significantly more aberrantly expressed gene population compared to the Sham group, while some aberrantly expressed genes showed a reversion trend after BC treatment. Figure 4 B-4C indicates that BC can partially reverse ICH-induced transcriptional abnormalities.

[0057] GO enrichment analysis showed that differentially expressed genes related to BC intervention mainly involved chemokine activity, leukocyte chemotaxis, neutrophil chemotaxis, macrophage chemotaxis, regulation of immune effector processes, receptor regulatory activity, extracellular matrix, platelet α-granules, and secretory granule cavities. Figure 4 D). KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in complement and coagulation cascades, phagocytosis, neuroactive ligand-receptor interactions, NETs formation, ECM-receptor interactions, integrin-related pathways, cytokine-cytokine receptor interactions, cell adhesion molecule interactions, and Toll-like receptor signaling pathways, etc. Figure 4 E).

[0058] The differential gene-pathway chord diagram shows that various inflammation, adhesion, and NETs-related genes are the main pathways involved in BC regulation. Figure 4 F).

[0059] GSEA results showed that, compared with the ICH group, BC treatment resulted in negative enrichment of multiple inflammation and immune-related pathways, including complement and coagulation cascades, NETs formation, ECM-receptor interactions, integrin signaling, cytokine-cytokine receptor interactions, cell adhesion molecule interactions, and Toll-like receptor signaling pathways. Among these, complement and coagulation cascades, integrin signaling, cell adhesion molecule interactions, and Toll-like receptor signaling pathways all showed significant negative enrichment. Figure 4 G).

[0060] The Venn plot results showed that there were 127 overlapping differential genes between the ICH vs Sham and BC vs ICH groups. Figure 4 H). The circular heatmap further shows that these overlapping genes exhibit significant aberrant expression after ICH treatment, while the expression of some genes shows a reverse regulatory trend after BC treatment ( Figure 4 I). These overlapping genes include inflammatory factors, chemokines, complement-related molecules, cell adhesion, and extracellular matrix-related genes.

[0061] The above results indicate that the DPP1 inhibitor BC can significantly regulate the transcriptome changes in brain tissue after cerebral hemorrhage and inhibit inflammatory and immune-related pathways such as complement coagulation, NETs formation, cell adhesion, integrin signaling, Toll-like receptor signaling, and chemokine activity.

[0062] 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. Application of dipeptidyl peptidase-1 inhibitors in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

2. The application according to claim 1, characterized in that, The dipeptidyl peptidase-1 inhibitor is Brensocatib or a pharmaceutically acceptable salt thereof.

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

4. The application according to claim 3, characterized in that, The secondary brain injury includes one or more of the following: cerebral edema, impaired hematoma absorption, blood-brain barrier disruption, neuronal degeneration or death, inflammatory cell infiltration, neutrophil extracellular trap-related damage, microglia / macrophage activation, or neurological deficits.

5. The application according to claim 1, characterized in that, The drug is used to achieve at least one of the following effects: reducing cerebral edema, promoting hematoma absorption, reducing neuronal damage, improving neurological deficits, or reducing inflammatory cell infiltration in brain tissue.

6. The application according to claim 1, characterized in that, The cerebral hemorrhage is selected from one of the following: spontaneous cerebral hemorrhage, hypertensive cerebral hemorrhage, basal ganglia hemorrhage, lobar hemorrhage, cerebellar hemorrhage, brainstem hemorrhage, or deep cerebral hemorrhage.

7. The application according to claim 1, characterized in that, The medication is administered within 72 hours of the onset of cerebral hemorrhage.

8. The application according to claim 7, characterized in that, When the subject is a human, the dosage of the dipeptidyl peptidase-1 inhibitor is 0.01 to 2.00 mg / kg body weight.

9. The application according to claim 8, characterized in that, The drug is administered once daily for 5-14 consecutive days.

Images