Cerebral stroke treatment time window extender, application thereof and cerebral stroke thrombolysis kit

Abstract

The invention relates to a cerebral apoplexy treatment time window prolonging agent, application of the cerebral apoplexy treatment time window prolonging agent and a cerebral apoplexy thrombolysis kit, and the cerebral apoplexy treatment time window prolonging agent comprises a protein PAI-1 targeting inhibitor, the compound is used for being specifically combined with a plasminogen activator inhibitor PAI-1 released by activated platelets in a cerebral apoplexy thrombus area so as to inhibit the fibrinolytic inhibitory activity of the PAI-1. The invention has the beneficial effects that endogenous fibrinolysis is activated in a safe and effective way, the sharp contradiction between timeliness and safety in the prior art is directly solved, and the medicine is expected to be used as a safe early intervention medicine and provides new treatment possibility for a large number of patients who miss initial treatment opportunities. By means of the exquisite unlocking mechanism, the cerebral apoplexy treatment time window is delayed to 6 h, and precious time is gained for cerebral apoplexy treatment subsequently.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a time window prolonging agent for stroke treatment and its application, and a thrombolytic reagent kit for stroke. Background Technology

[0002] Stroke is a leading cause of death and disability, with acute ischemic stroke (AIS) accounting for the majority. For AIS, early reperfusion of the occluded vessel is crucial for improving prognosis. In clinical treatment of stroke, intravenous thrombolytic drugs (such as recombinant tissue plasminogen activator rt-PA, brand name alteplase) are widely used. However, thrombolytic drugs are limited by strict dosing windows and the risk of bleeding complications such as intracranial hemorrhage, resulting in a limited proportion of patients who can benefit. Furthermore, in newly formed thrombi, the endogenous fibrinolytic inhibitor PAI-1 can rapidly inhibit the activity of plasminogen activator tPA (tissue plasminogen activator) and uPA (urokinase plasminogen activator), with significantly elevated concentrations in the thrombus and platelet-activated environment, easily leading to thrombolytic resistance and decreased recanalization rates. Clinically, higher doses of thrombolytic drugs are often required to counteract the inhibitory effect; however, this further increases the risk of bleeding.

[0003] Existing research has shown that specific inhibition of PAI-1 can effectively reduce the therapeutic dose of alteplase, thereby significantly reducing the risk of cerebral hemorrhage while maintaining thrombolytic efficacy (Xu Y. et al., "Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment", International Journal of Biological Macromolecules, 2024, 257:128618). However, due to the limited time window for thrombolytic drug administration, delays in pre-hospital transport or delayed arrival at the hospital for patients with acute ischemic stroke will directly lead to missing or significantly compressing the time window for intravenous thrombolysis. This not only deprives patients of the opportunity to receive the most effective early reperfusion therapy, but also accelerates the transformation of the ischemic penumbra into irreversible infarction, significantly increasing disability and mortality rates, and exacerbating the medical and social burden. Summary of the Invention

[0004] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a stroke treatment time window prolonging agent and its application, and a stroke thrombolysis kit, which solves the technical problem that the stroke treatment time window is limited, thus affecting the subsequent stroke treatment effect.

[0005] (II) Technical Solution To achieve the above objectives, the main technical solutions adopted by the present invention include: In a first aspect, embodiments of the present invention provide a stroke treatment time window extension agent, including a protein-based PAI-1 targeting inhibitor, for specifically binding to plasminogen activator inhibitor PAI-1 released by activated platelets in the stroke thrombus region to inhibit the fibrinolytic inhibitory activity of PAI-1.

[0006] Stroke treatment time window extenders are used in the emergency phase to administer medication to suspected stroke cases in order to extend the treatment time window for confirmed stroke cases. The treatment time window is the period from the onset of stroke to the administration of thrombolytic therapy after diagnosis.

[0007] In a preferred embodiment of the present invention, the stroke treatment time window prolonging agent is wherein the protein-based PAI-1 targeting inhibitor is PAITrap4, which is modified with the platelet-targeting peptide cRGD to specifically bind to PAI-1 released by activated platelets in the stroke thrombus region.

[0008] Secondly, embodiments of the present invention provide the use of protein PAI-1 targeting inhibitors in the preparation of pharmaceutical compositions or kits for prolonging the time window of thrombolytic therapy for ischemic stroke.

[0009] In a preferred embodiment of the present invention, the drug composition or kit is administered via intravenous injection.

[0010] In a preferred embodiment of the present invention, the pharmaceutical composition or kit is a dosage form for intravenous injection.

[0011] In a preferred embodiment of the present invention, the protein-based PAI-1 targeting inhibitor described herein is PAITrap4.

[0012] In a preferred embodiment of the present invention, the protein-based PAI-1 targeting inhibitor is further modified with the platelet-targeting peptide cRGD.

[0013] Thirdly, embodiments of the present invention provide a kit for thrombolysis in ischemic stroke, comprising a first preparation and a second preparation packaged separately, wherein the first preparation comprises PAItrap4 and the second preparation comprises plasminogen activator.

[0014] In this kit, the first preparation is administered before the second preparation. The first preparation can be administered as soon as stroke symptoms are detected; the second preparation is administered 3-6 hours after the stroke occurs.

[0015] In the first formulation, the dosage of PAItrap4 is 5-20 mg / kg, preferably 8-12 mg / kg; in the second formulation, the dosage of plasminogen activator (e.g., alteplase) is 3-10 mg / kg, preferably 4-6 mg / kg.

[0016] In a preferred embodiment of the present invention, the plasminogen activator in the kit is alteplase.

[0017] Fourthly, embodiments of the present invention provide the application of a drug kit of PAItrap4 and alteplase in the preparation of thrombolytic agents for ischemic stroke.

[0018] In the thrombolytic agents for ischemic stroke mentioned here, PAItrop4 and alteplase are administered sequentially, with an interval of 3-6 hours.

[0019] (III) Beneficial Effects The beneficial effects of this invention are as follows: The stroke treatment time window extension agent and its application, as well as the stroke thrombolysis kit of this invention, utilize a protein-based PAI-1 inhibitor (PAItrap4). Its core mechanism of action is not direct thrombolysis, but rather efficient neutralization of the key molecule PAI-1, which causes "thrombolysis resistance," at the lesion site. This relieves PAI-1's inhibition of the body's own tPA / uPA activity, restoring and enhancing the activity of the endogenous fibrinolytic system. By activating endogenous fibrinolysis in a safe and effective manner, this invention directly resolves the sharp contradiction between timeliness and safety in existing technologies. It holds promise as a safe early intervention drug, providing new treatment possibilities for a large number of patients who have missed the initial treatment opportunity. Compared to existing technologies, this ingenious "unlocking" mechanism extends the stroke treatment time window to 6 hours, gaining valuable time for subsequent stroke treatment.

[0020] Furthermore, PAITrap4 is a high-affinity PAI-1 inhibitor fused to the platelet-targeting peptide cRGD, enabling it to specifically accumulate in platelet-rich thrombus regions. In a mouse model of carotid artery embolism (CES) ischemic stroke, PAITrap4 injection successfully extended the effective treatment window of alteplase from the traditional 4.5 hours to 6 hours, and even with delayed treatment, it still significantly promoted cerebral blood flow restoration, reduced infarct volume, and improved neurological function scores.

[0021] Furthermore, PAItrap4 pretreatment significantly enhanced endogenous fibrinolytic activity (plasma D-dimer levels in ischemic stroke mice increased from 260.6±60.5 ng / mL to 610.0±65.9 ng / mL) and reduced brain tissue inflammatory factors TNF-α and IL-6 levels, as well as blood-brain barrier permeability (Evans Blue exudate decreased from 21.5±1.1 μg / g to 7.1±1.4 μg / g), suggesting that it helps protect vascular integrity and reduce the risk of hemorrhagic transformation while prolonging the time window of thrombolytic therapy.

[0022] More importantly, this invention significantly improves safety by activating a milder endogenous pathway. Alteplase is a plasminogen activator whose main function is to activate the conversion of plasminogen activator (PLG) to plasminogen activator (PLN). However, PLN is a non-specific enzyme, and its excessive activity can lead to cerebral hemorrhage. PAItrap4, as an inhibitor of PAI-1, has platelet-targeting properties, locally enhancing fibrinolytic activity within the thrombus area while avoiding systemic increases in the non-specific activity of PLN, thereby reducing the risk of cerebral hemorrhage caused by excessively high local PLN concentrations. In addition, PAItrap4 can also reduce the risk of cerebral hemorrhage by inhibiting PAI-1, improving vascular permeability, mitigating reperfusion injury, and enhancing microvascular stability. In the mouse hemorrhagic stroke (ICH) model, where safety requirements are most stringent, injection of PAItrap4 not only did not aggravate cerebral hemorrhage but also showed a trend of reducing cerebral edema and blood-brain barrier disruption, demonstrating excellent safety. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the process of activating endogenous fibrinolytic activity by injecting PAItrap4, a time window extender for stroke treatment. Figure 2 To verify the evaluation of the effect of PAItrap4 pretreatment on alteplase delayed thrombolysis in mice with ischemic stroke in Example 1; where, a is a schematic diagram of the experimental grouping, time axis and dosing regimen; b is a representative image of regional cerebral blood flow (rCBF) monitored by two-dimensional laser speckle imaging (2D-LSCI), showing the blood flow recovery at 2 hours (+2 hours) and 4 hours (+4 hours) after alteplase treatment at different delay times; c is a quantitative statistical graph of the changes in regional cerebral blood flow (rCBF) corresponding to Figure B; Figure 3To verify the assessment results of PAItrap4 pretreatment on neurological deficits, infarct volume, and brain tissue damage in mice with ischemic stroke in Example 2, the following figures are presented: a) is a statistical graph of neurological deficit scores based on the Longa Scale for each group of mice; b) is a representative image of TTC-stained coronal sections of brain tissue from each group of mice; c) is a quantitative statistical graph of infarct volume from each group of mice corresponding to Figure B; d) is a HE-stained pathological section of brain tissue from the corresponding region of the ischemic hemisphere in each group of mice, with arrows indicating vacuolar degeneration and irregular, condensed, and deeply stained cell nuclei. Figure 4 The figure shows the results of the measurement of D-dimer concentration in the plasma of each group of CES mice in Example 3 after 24 hours of treatment with saline or PAItrap4. Figure 5 To verify the detection results of PAItrap4 on cerebral hemorrhage in mice with ischemic stroke in Example 4; where a is an image of the supernatant of the homogenate of the left (ischemic) hemisphere tissue of each group of mice; b is a quantitative statistical graph of the amount of cerebral hemorrhage detected by spectrophotometric hemoglobin assay. Figure 6 To verify the results of PAItrap4's assessment of the level of inflammation and cerebral vascular integrity in the brain tissue of mice with ischemic stroke in Example 5, the following figures are shown: a and b are the results of the measurement of the levels of inflammatory factors TNF-α and IL-6 in the brain tissue of mice in each group, respectively; c is a quantitative statistical graph of the Evans Blue content in the brain tissue of mice in each group; d is a representative fluorescence image of FITC-glucan perfusion in the microvessels of the ischemic area of ​​mice in each group, with red arrows indicating the leakage areas of FITC-glucan. Figure 7 To verify the evaluation results of PAItrap4 on brain injury and neurological function in hemorrhagic stroke (ICH) mice in Example 6, the following graphs are presented: a) is a quantitative statistical graph of the amount of hemorrhage in the brain tissue of each group of ICH mice detected by spectrophotometric hemoglobin assay; b) is a graph of the Evans blue content in the brain tissue of each group of ICH mice; c) is a graph of the water content in the ipsilateral and contralateral cerebral hemispheres of each group of ICH mice; d) is a representative section of Prussian blue stained brain tissue of each group of ICH mice, with black arrows indicating the iron ions (blue) formed after erythrocyte lysis; e) is a statistical graph of the neurological deficit scores of each group of ICH mice based on the Longa scale within 10 hours after modeling. Detailed Implementation

[0024] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0026] First, let's explain the abbreviations and key terms: PAItrap4: A high-affinity PAI-1 inhibitor, PAItrap4 is fused to the C-terminus of HSA with a cyclic RGD peptide (see paper Xu Y. et al., "Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment", International Journal of Biological Macromolecules, 2024, 257: 128618).

[0027] PAI-1: Plasminogen activator inhibitor-1, belongs to the serine protease inhibitor family (serpin family), and is one of the most important fibrinolytic inhibitors in the body.

[0028] CES: A mouse model of ischemic stroke induced by carotid artery embolism.

[0029] ICH: Hemorrhagic mouse stroke model.

[0030] LSCI: Laser speckle flow imaging.

[0031] CBF: Cerebral blood flow. CBF indirectly reflects the flow velocity of red blood cells in blood vessels on the surface of brain tissue by analyzing the contrast changes in speckle images.

[0032] PLG: Plasminogen, an inactive precursor that is converted into plasmin after activation.

[0033] PLN: Plasmin, derived from plasminogen (PLG) activated by tPA, is a key protease responsible for directly degrading the fibrin network structure, thus playing a role in dissolving blood clots.

[0034] Saline: physiological saline solution.

[0035] See Figure 1The flowchart illustrates the activation of endogenous fibrinolytic activity by PAItrap4, an injection agent that extends the treatment time window for stroke. PAItrap4 can be administered as soon as stroke symptoms appear. Pre-injection during the emergency phase (in the ambulance) inhibits PAI-1 activity, releasing tPA / uPA and activating endogenous plasmin. After diagnosis to determine the type of stroke (hemorrhagic or ischemic stroke), PAItrap4 has no side effects in hemorrhagic stroke. In ischemic stroke, PAItrap4 prolongs the thrombolytic therapy time window.

[0036] Example 1 This embodiment provides an application of a time window extender for stroke treatment. A carotid embolism-induced ischemic stroke animal model is used. After modeling, PAItrap4 is administered as a lead drug at a dose of 10 mg / kg (PAItrap4 group, prepared with sterile PBS), or normal saline is administered as a lead drug at a dose of 10 mg / kg (Saline group); then, as... Figure 2 As shown in Figure a, alteplase was administered intravenously to the PAItrap4 group and the Saline group at a dose of 5 mg / kg for thrombolysis 3 or 6 hours after modeling, resulting in four experimental groups: Saline + Alteplase 3 rd hour group, PAItrap4+Alteplase3 rd hour group, Saline + Alteplase 6 th hour group and PAItrarap4+Alteplase 6 th hour group.

[0037] Verification Example 1 This validation example used LSCI to monitor cerebral blood flow in mice of each treatment group in Example 1 and compared changes in blood flow in the stroke hemisphere before and after treatment. The results are as follows: Figure 2 As shown in Figures b and c, there was no significant difference in blood flow among the CES mice in each group after modeling, indicating that the degree of ischemia was consistent across all groups. Among the CES mice treated with alteplase 3 hours after modeling: mice pre-injected with Saline saline (Saline + Alteplase 3...) rd In the alteplase group, blood flow recovered to 42.2% and 44.3% of baseline at 2 h and 4 h, respectively; however, in mice pre-injected with PAItrap4 (PAItrap4 + Alteplase 3), blood flow recovered to baseline. rd hour group), corresponding to Saline + Alteplase 3 rdAt the same time points in the hour group, blood flow recovered to 63.8% and 84.1% of baseline, respectively, compared to Saline + Alteplase 3. rd Compared to the hour group: +2 h: p<0.05; +4 h: p<0.0001.

[0038] In CES mice treated with Alteplase 6 hours after successful modeling: Mice pre-injected with Saline (Saline + Alteplase 6) th In the alteplase group, blood flow recovered to 28.9% of baseline 2 hours after alteplase treatment and reached 36.2% after 4 hours. However, in mice pre-injected with PAItrap4 (PAItrap4+Alteplase), blood flow was significantly reduced. 6th At the same time point (hour), blood flow recovered to 57.2% and 67.3% of baseline, respectively, compared to Saline + Alteplase 6. th Compared to the +2 h group: p<0.01; +4 h: p<0.0001. These results indicate that pre-injection of PAItrap4 significantly accelerated the rate of blood flow recovery in CES mice, whether alteplase treatment was delayed by 3 h or 6 h.

[0039] Verification Example 2 This validation case evaluated the recovery of neurological function, cerebral infarction, and brain tissue damage in CES mice after alteplase thrombolysis treatment at 3 h and 6 h post-modeling. Results are shown below. Figure 3 .

[0040] Statistical results of neurological function scores are as follows Figure 3 As shown in Figure a, in CES mice treated with alteplase 3 h after modeling: mice pre-injected with Saline saline (Saline + Alteplase 3) rd In the hour group, the score decreased from 3.7 to 2.8 points 1 to 4 hours after alteplase treatment, a smaller decrease. However, mice pre-injected with PAItrap4 (PAItrap4 + Alteplase 3) showed a smaller decrease. rd In the hour group, from 1 to 4 hours after receiving alteplase treatment, the score dropped from 3.5 to 0.8, a significant decrease, and the behavioral performance improved from being unable to move independently to being able to walk freely.

[0041] In CES mice treated with alteplase 6 hours after modeling: mice pre-injected with Saline (Saline + Alteplase 6) thIn the hourly group, mice treated with alteplase maintained a score above 3 for 4 hours, were unable to walk independently, and leaned to the left. However, mice pre-injected with PAItrap4 (PAItrap4+Alteplase) showed significantly better results. 6th Two hours after receiving alteplase treatment, the mouse score dropped from 3.6 to 2.7. Although the mouse was conscious, it only had a slight ability to walk. Four hours after treatment, the score dropped to around 1.3, and the mouse was able to walk spontaneously, indicating a significant improvement in neurological function. These results demonstrate that pre-injection of PAItrap4 significantly enhances the neurological function recovery effect of delayed alteplase treatment in the CES mouse model.

[0042] TTC staining was performed on the brain tissues of each treatment group in Example 1, such as... Figure 3 As shown in Figure b, the corresponding quantitative statistics of cerebral infarction volume are as follows: Figure 3 The image shows that in CES mice treated with alteplase 3 hours after modeling: mice pre-injected with Saline saline (Saline + Alteplase 3) rd The percentage of infarct volume in the hour group (15.6 ± 1.1%) was higher than that in mice pre-injected with PAItrap4 (PAItrap4 + Alteplase 3). rd hour group (2.0±0.5%). Furthermore, in CES mice treated with alteplase 6 h after modeling: mice pre-injected with Saline (Saline + Alteplase 6 h) th The percentage of infarct volume in the hour group (24.9 ± 2.6%) was significantly higher than that in mice pre-injected with PAItrap4 (PAItrap4 + Alteplase). 6th The results indicate that pre-injection of PAItrap4 can reduce brain tissue damage induced by delayed alteplase treatment and decrease the infarct volume in CES mice (1.9 ± 0.5%).

[0043] Assess the effect of pre-injection of PAItrap4 on brain tissue damage induced by delayed alteplase treatment in CES mice. Figure 3As shown in Figure d, HE-stained sections revealed significant morphological differences in lesion development between sham-operated mice (Sham group, which underwent the same surgical exposure procedures as CES modeling, including anesthesia, neck incision, and vascular dissection, but without electrical stimulation to form thrombi or pushing them into intracranial vessels to cause occlusion) and CES mice. Neuronal cell structure in sham-operated mice was normal, with clear margins and homogeneous staining of nuclei and intercellular matrix. However, brain tissue from CES mice and mice treated with Saline and alteplase showed vacuolar necrosis, widened intercellular spaces, intercellular edema, and blurred cell boundaries. Nuclei exhibited irregular, pyknotted, and deeply stained changes. The longer the alteplase treatment was delayed, the more severe the brain tissue damage. However, combined treatment with PAItrap4 and alteplase alleviated brain damage to varying degrees, including a reduction in neuronal necrosis and intercellular edema. More importantly, these improvements were almost unaffected by the delay in alteplase treatment.

[0044] Verification Example 3 This validation example assesses the activation effect of PAItrap4 on the endogenous fibrinolytic system by detecting D-dimer levels, thus verifying its mechanism of action. D-dimer is a specific degradation product of cross-linked fibrin, and its elevated level reflects enhanced secondary fibrinolytic activity. Immediately after CES modeling, mice were injected via tail vein with PAItrap4 (10 mg / kg) or saline as a control. Plasma was collected 24 hours later, and D-dimer concentration was detected using ELISA. Results are as follows... Figure 4 As shown, in the PAIrap4 treatment group, the D-dimer concentration in mouse plasma reached 610.0 ± 65.9 ng / mL, which was significantly increased compared to 260.6 ± 60.5 ng / mL in ischemic stroke mice treated with saline alone (p < 0.0001). This result provides direct biochemical evidence that PAIrap4 not only passively inhibits PAI-1, but also significantly enhances the intrinsic fibrinolytic activity of mice by inhibiting PAI-1, thereby maintaining effective thrombolytic capacity even with delayed alteplase administration.

[0045] Verification Example 4 This validation case evaluated the effect of PAItrap4 on the risk of hemorrhage in CES mice. Under different treatment conditions (including the Saline group without alteplase and the groups treated with alteplase at 3 or 6 hours post-modeling), the amount of hemorrhage in the mouse brain tissue was measured within 4 hours after the corresponding treatment. Results are shown below. Figure 5 .

[0046] like Figure 5Results a and b show that in CES mice treated with alteplase 3 hours after modeling: CES mice pre-injected with PAItrap4 (PAItrap4 + Alteplase 3) rd The amount of cerebral hemorrhage in the hour group (0.67±0.02 μL) was lower than that in CES mice treated with Saline alone (Saline+Alteplase 3). rd The amount of cerebral hemorrhage in the pre-treated group (6 hours after modeling) was 0.76 ± 0.05 μL, significantly lower than that in mice pre-injected with Saline (0.83 ± 0.04 μL; p < 0.05). This advantage was even more pronounced in CES mice treated with alteplase 6 hours after modeling. In CES mice treated with alteplase 6 hours after modeling, the amount of hemorrhage in mice pre-injected with Saline (1.06 ± 0.03 μL) was significantly higher than that in mice pre-injected with PAItrap4 (0.63 ± 0.05 μL; p < 0.01). This indicates that pre-injection of PAItrap4 can significantly reduce the amount of cerebral hemorrhage in CES mice treated with delayed alteplase.

[0047] Verification Example 5 This validation case evaluated the effects of PAItrap4 on the level of brain inflammation and cerebral vascular integrity in CES mice with ischemic stroke. Immediately after CES modeling, PAItrap4 (10 mg / kg) or saline was injected via the tail vein. Brain tissue was harvested 24 hours later to detect the levels of inflammatory factors TNF-α and IL-6. Blood-brain barrier permeability and microvascular perfusion in the ischemic area were assessed by Evans blue extravasation and FITC-glucan perfusion. Results are shown below. Figure 6 .

[0048] The results are as follows Figure 6 As shown in Figures a and b: Compared with the saline group, the TNF-α level in brain tissue of the PAItrap4 treatment group decreased from 2.0±0.3 ng / g to 1.3±0.1 ng / g (p<0.05), and the IL-6 level decreased from 12.3±4.9 ng / g to 1.1±0.2 ng / g (p<0.05); Figure 6 As shown in Figure C, the Evans Blue content in brain tissue decreased from 21.5 ± 1.1 μg / g to 7.1 ± 1.4 μg / g (p < 0.01). See also... Figure 6 The FITC-glucan perfusion results showed that the saline group had insufficient microvascular perfusion and significant leakage in the ischemic area, while the PAItrap4 group had more complete microvascular perfusion and reduced leakage. These results indicate that PAItrap4 can reduce the inflammatory response after ischemic stroke and protect the integrity of cerebral blood vessels, thereby helping to reduce the risk of hemorrhagic transformation.

[0049] Verification Example 6 This validation case evaluated the safety of PAItrap4 in a mouse model of hemorrhagic stroke (ICH) and its effects on brain tissue damage and neurological function. An ICH model was established using autologous blood injection (20 μL of autologous blood injected into the left basal ganglia). Immediately after modeling, PAItrap4 (10 mg / kg) or an equal volume of saline was injected via the tail vein as a control. 24 h after administration, brain hemorrhage volume, blood-brain barrier permeability, brain water content, Prussian blue-positive iron deposition, and neurological deficit scores within 10 h were measured. Results are shown below. Figure 7 .

[0050] like Figure 7 As shown, compared with the saline group, the amount of cerebral hemorrhage in the PAItrap4 treatment group was significantly reduced ( Figure 7 (a, P<0.01; n=6 / group); Evans Blue, a blood-brain barrier permeability indicator, was exuded / concentrated with decreased levels. Figure 7 (b, P<0.05; n=12 / group). Regarding cerebral edema, the water content of the ipsilateral (injured side) brain in the saline group was significantly higher than that on the contralateral side. Figure 7 (c, P<0.001), while there was no significant difference in ipsilateral and contralateral brain water content after PAItrap4 treatment. Meanwhile, compared with the saline group, PAItrap4 significantly reduced ipsilateral brain water content (c <0.001). Figure 7 c, P<0.05; n=10 / group). Prussian blue staining showed a decrease in positive signal / positive cells in PAItrap4 group ( Figure 7 (d, n=3 / group). Furthermore, the neurological deficit scores in the PAItrap4 group decreased more rapidly over time, suggesting that it can promote neurological function recovery. Figure 7 (e, n=10 / group). In summary, PAItrap4 did not aggravate cerebral hemorrhage, blood-brain barrier disruption, or cerebral edema in the ICH model, suggesting that its pre-administration during the emergency phase has good safety.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A time window prolonging agent for stroke treatment, characterized in that, Including protein-based PAI-1 targeting inhibitors, which specifically bind to plasminogen activator inhibitors (PAI-1) released by activated platelets in the thrombus region of stroke to inhibit the fibrinolytic activity of PAI-1.

2. The stroke treatment time window prolonging agent as described in claim 1, characterized in that, The protein-based PAI-1 targeting inhibitor is PAITrap4, which is modified with the platelet-targeting peptide cRGD to specifically bind to PAI-1 released by activated platelets in the thrombus region of stroke.

3. Application of protein PAI-1 targeting inhibitors in the preparation of pharmaceutical compositions or kits for prolonging the time window of thrombolytic therapy for ischemic stroke.

4. The application as described in claim 3, characterized in that, The route of administration for the pharmaceutical composition or kit is intravenous injection.

5. The application as described in claim 4, characterized in that, The pharmaceutical composition or kit is a dosage form for intravenous injection.

6. The application as described in claim 3, characterized in that, The protein-based PAI-1 targeting inhibitor is PAITrap4.

7. The application as described in claim 3 or 6, characterized in that, The protein-based PAI-1 targeting inhibitor is also modified with the platelet-targeting peptide cRGD.

8. A kit for thrombolysis in ischemic stroke, characterized in that, It includes a first formulation and a second formulation packaged separately. The first formulation includes PAItrap4, and the second formulation includes plasminogen activator.

9. The reagent kit as described in claim 8, characterized in that, The plasminogen activator is alteplase.

10. Application of the PAItrap4 and alteplase drug kit in the preparation of thrombolytic agents for ischemic stroke.

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