Application of elastase inhibitors in the preparation of drugs for treating cerebral hypoperfusion

Abstract

The present disclosure relates to the field of medicine, and more particularly to the use of elastase inhibitors in the preparation of medicaments for treating cerebral hypoperfusion. Studies in the disclosed examples have revealed that NE expression is elevated in the corpus callosum of patients with cerebral hypoperfusion. NE can cause damage to oligodendrocytes and white matter lesions. Elastase inhibitors can effectively inhibit NE expression and activity, reducing oligodendrocyte damage. They also mitigate the enzymatic cleavage of NE on the myelin protein CNPase, reducing myelin damage in the corpus callosum and thereby inhibiting white matter lesions.

Description

Technical Field

[0001] The present disclosure relates to a new medical application field of elastase inhibitors, and in particular to the application of elastase inhibitors in the preparation of drugs for treating cerebral hypoperfusion. Background Art

[0002] White matter lesion (WML) caused by cerebral hypoperfusion can impair sensorimotor and cognitive functions and trigger oligodendrocyte apoptosis and myelin degeneration. Neutrophil elastase (NE) is a serine protease that degrades foreign microorganisms or organic molecules phagocytosed by neutrophils. However, brain injury causes neutrophils to enter the brain tissue and release NE, which degrades various structurally and functionally important molecules in the brain tissue, disrupting the blood-brain barrier and participating in the inflammatory response to brain injury, inducing neuronal death, and mediating inflammation-related secondary glial scar formation in spinal cord injury models. However, the pathogenesis of white matter lesions and cognitive dysfunction caused by cerebral hypoperfusion is currently poorly understood, which greatly hinders the development of new therapeutic drugs. Therefore, it is urgent to find new mechanisms that cause oligodendrocyte injury and white matter lesions, as well as to develop related applications of existing drugs. Summary of the Invention

[0003] Elastase inhibitors inhibit the activity and production of elastase. Currently, elastase inhibitors, especially neutrophil elastase inhibitors, have demonstrated tissue protective effects in lung injury, pancreatitis, and nephritis. However, their use in the treatment of injuries caused by cerebral hypoperfusion has not been reported. To address the above technical issues, the present disclosure provides the use of elastase inhibitors in the preparation of medicaments for treating cerebral hypoperfusion.

[0004] The present disclosure provides the use of an elastase inhibitor in the preparation of a medicament for treating brain white matter damage and / or oligodendrocyte damage.

[0005] The present disclosure provides the use of an elastase inhibitor in the preparation of a medicament for treating vascular dementia.

[0006] As a preferred technical solution of the present disclosure, an elastase inhibitor is used in the preparation of a drug for inhibiting the enzymatic degradation of myelin protein CNPase induced by NE.

[0007] As a preferred technical solution of the present disclosure, the elastase inhibitor includes sivelestat.

[0008] As a preferred technical solution of the present disclosure, the preparation of the drug is a lyophilized powder or an injection.

[0009] As a preferred technical solution of the present disclosure, the preparation further includes one or more pharmaceutically acceptable carriers or excipients.

[0010] As a preferred technical solution of the present disclosure, the pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives.

[0011] The technical solution provided by the embodiments of the present disclosure has the following advantages over the prior art:

[0012] The disclosed examples have been studied and found that the expression of NE in the corpus callosum of patients with cerebral ischemia is elevated, and NE can cause brain oligodendrocyte damage and white matter lesions. Elastase inhibitors can effectively inhibit the expression and activity of NE, reduce oligodendrocyte damage, and at the same time reduce the enzymatic cleavage of NE on the myelin protein CNPase, reduce myelin damage in the corpus callosum, and thus inhibit white matter lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0014] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0015] Figure 1 The NE in the corpus callosum of one of the human specimens in the control group and one of the patients with cerebral small vessel disease (CSVD) is shown in the embodiment of the disclosure. + Staining images of cells.

[0016] Figure 2 for Figure 1 NE in the corpus callosum of human specimens in the control group + Cells and Figure 1 NE in the corpus callosum of patients with midbrain small vessel disease (CSVD) + Quantitative plots of cells.

[0017] Figure 3In the examples of the present disclosure, NE protein concentration in the corpus callosum of sham-operated control mice and CSVD white matter damage-induced bilateral common carotid artery stenosis (BCAS) mice was assessed using enzyme-linked immunosorbent assay at different time points.

[0018] Figure 4 This is a graph showing the NE catalytic activity in corpus callosum homogenate samples from sham-operated control mice and BCAS-induced CSVD white matter damage mice at different time points using spectrophotometry in the embodiments of the present disclosure.

[0019] Figure 5 Representative optical microscopy images and quantitative graphs of active cells after oligodendrocyte precursor cells (OPCs) were treated with different concentrations of NE in the examples of the present disclosure.

[0020] Figure 6 Representative optical microscopy images and quantitative graphs of active cells after OPC cells were treated with 40 nmol / L NE at different time points in the examples of the present disclosure.

[0021] Figure 7 In the embodiment of the present disclosure, A2B5 positively labeled OPC cells were treated with solvent and NE for 5 hours, and caspase-3 + (CC3 + ) Representative confocal images and quantitative plots of cells.

[0022] Figure 8 The graph is a quantitative graph of active cells in differentiated mature oligodendrocytes treated with different concentrations of NE according to the examples of the present disclosure.

[0023] Figure 9 In the embodiment of the present disclosure, CC3 in differentiated mature oligodendrocytes treated with solvent and NE were used. + Representative confocal images and quantification of cells.

[0024] Figure 10 CNPase + Representative confocal images and quantitative graphs of cells in NG2-positive OPC cells.

[0025] Figure 11 Representative confocal images of mature oligodendrocytes differentiated from the present invention were treated with solvent and NE for 5 hours, respectively, and then labeled with MBP and CNPase, as well as quantitative graphs of the mean fluorescence intensity of CNPase staining.

[0026] Figure 12This is a diagram of the domain structure of the CNPase protein according to the disclosed embodiment.

[0027] Figure 13 This is a diagram of the enzymatic cleavage products of recombinant human CNPase after being cleaved by human natural NE under different enzyme and substrate (i.e., NE and CNPase) mass ratios (right) and different enzymatic cleavage times (left) as evaluated by SDS-PAGE and Coomassie Brilliant Blue staining in the embodiments of the present disclosure.

[0028] Figure 14 This is a map of the NE cleavage site and sequence amino acid conservation in the human CNPase protein and mouse CNPase protein of the embodiments of the present disclosure.

[0029] Figure 15 These are representative images of the mouse corpus callosum stained with MBP, LFB, and Olig2, respectively, in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the disclosed embodiments.

[0030] Figure 16 30 days after surgery, the quantitative graph shows the mean fluorescence intensity of MBP in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group.

[0031] Figure 17 30 days after surgery, the figures show the extent of white matter lesions stained by LFB in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group in the examples of the present disclosure.

[0032] Figure 18 The expression of Olig2 in the corpus callosum of the mice in the sham operation group, the model group solvent administration control group, the model group low-dose administration group and the model group high-dose administration group 30 days after surgery in the disclosed embodiment was as follows: + Quantitative plots of cells.

[0033] Figure 19 Representative electron micrographs of myelin sheaths in the corpus callosum of mice in the sham-operated control group, the model solvent-administered control group, the model low-dose administration group, and the model high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0034] Figure 20 This is a quantitative analysis graph of the g-ratio values ​​of nerve fibers (axons + myelin sheaths) in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0035] Figure 21This is a scatter plot of the functional relationship between the g-ratio value and the axon diameter of the mice in the sham operation control group, the model group solvent administration control group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0036] Figure 22 This is a graph showing the axon diameters of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0037] Figure 23 Graphs showing the results of the rotarod fatigue test of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group at different time points in the disclosed embodiments.

[0038] Figure 24 28 days after surgery, the results of the object recognition test were performed on mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group in the examples of the present disclosure.

[0039] Figure 25 This is a graph showing the results of a Y-maze test on mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 29 days after surgery in the examples of the present disclosure. DETAILED DESCRIPTION

[0040] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features therein can be combined with each other in the absence of conflict.

[0041] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0042] The disclosed embodiments propose the use of elastase inhibitors in the preparation of drugs for treating cerebral hypoperfusion. Studies have found that inhibiting NE through administration of elastase inhibitors can significantly increase the survival rate of oligodendrocyte lineage cells, reduce the progression of white matter damage, and provide lasting improvements in behavioral outcomes after hypoperfusion injury.

[0043] The second aspect of the disclosed embodiments further proposes the use of an elastase inhibitor in the preparation of a drug for treating brain white matter damage and / or oligodendrocyte damage.

[0044] The third aspect of the disclosed embodiments further proposes the use of an elastase inhibitor in the preparation of a drug for treating vascular dementia.

[0045] The fourth aspect of the present disclosure further proposes the use of an elastase inhibitor in the preparation of a drug for inhibiting the enzymatic degradation of myelin protein CNPase induced by NE.

[0046] As a preferred embodiment of the present disclosure, the elastase inhibitor includes sivelestat.

[0047] In some specific embodiments, the medicine is administered by a method comprising administering the medicine intravenously, intraperitoneally, intracoronaryally, intraarterially, intradermally, subcutaneously, transdermally, intratracheally, intraarticularly, intraventricularly, cranially, by suction, intracerebrally, orally, intraocularly, pulmonaryly, by catheter injection, by suppository, and directly by injection into tissue. In addition, coloring agents, preservatives, spices, flavoring agents, or other additives may be added to the pharmaceutical preparation as needed. To achieve the desired medication effect, the medicine or pharmaceutical composition of the present disclosure may be administered by any known method of administration.

[0048] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0049] Example:

[0050] 1. Human specimens and experimental methods

[0051] 1. Human specimens

[0052] Human sample collection was performed according to protocols approved by the Institutional Review Board of Beijing Tiantan Hospital (Beijing, China). For immunostaining of human brain tissue, three control brain sections and eight brain sections with cerebral small vessel disease (CSVD) were included. The control brain tissue was obtained from deceased individuals with non-neurological diseases and no history of neuropsychiatric disorders.

[0053] 2. Experimental methods

[0054] Human brain sections of 5 μm thickness were cut and immunostained with NE antibody (ab68672, purchased from Abcam). Five randomly selected microscopic fields in the corpus callosum of each brain slide were examined, and the corpus callosum 0.15 mm was counted. 2 NE in the field of vision +The researchers were unaware of the clinical diagnosis when examining the samples. There was no statistically significant difference in mean age between the CSVD group and the control group [CSVD group: 57.7 ± 2.19 years; control group: 61.8 ± 3.42 years; mean ± SEM; P > 0.05; unpaired t-test].

[0055] 2. Animal Experiments

[0056] 1. Experimental animals: Male C57BL / 6 mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.), 8 weeks old, were randomly divided into 4 groups, 7 mice in each group, namely:

[0057] Sham group: received sham surgery;

[0058] Model group: Solvent administration control group (BCAS+Vehicle): PBS buffer (Biosharp, cat No. BL302A, purchased from Beijing Lanjieke Technology Co., Ltd.) was administered after model establishment;

[0059] Model group low-dose administration group (BCAS+Sivelestat, L): After modeling, the rats were given a 1 mg / kg sivelestat drug preparation (ONO-5046, catalog #3535, purchased from Tocris Bioscience); where L stands for Low.

[0060] Model group high-dose administration group (BCAS+Sivelestat, H): After modeling, the drug preparation of Sivelestat at a concentration of 10 mg / kg was administered; wherein, H stands for High.

[0061] 2. Animal experimental methods:

[0062] 2.1BCAS model establishment:

[0063] Mice were anesthetized with 4.0% isoflurane and maintained on a 70% N₂O / 30% O₂ system using 1.5% isoflurane. A midline incision was performed on the mice's neck, exposing both common carotid arteries. Microcoils with a diameter of 0.18 mm (purchased from Wuxi Samini Spring Co., Ltd.) were surgically implanted into the common carotid arteries bilaterally. Rectal temperature was maintained between 36.5°C and 37.5°C.

[0064] 2.2 Sham operation method: The specific operation was the same as that of the experimental group, but the surgery only exposed both common carotid arteries and no microcoil was implanted.

[0065] 3. Dosage method:

[0066] Sham control group (Sham): no reagent was given;

[0067] Model group and solvent administration control group (BCAS+Vehicle): After successful model establishment, 100 μL of PBS buffer solution was administered intraperitoneally once a day for 4 weeks;

[0068] Model group low-dose administration group (BCAS + Sivelestat, L): 100 μL of 1 mg / kg sivelestat drug preparation was administered once 24 hours before BCAS surgery; after successful model establishment, 100 μL of 1 mg / kg sivelestat drug preparation was administered once daily, both by intraperitoneal injection, for 4 weeks;

[0069] Model group high-dose administration group (BCAS+Sivelestat, H): 100 μL of 10 mg / kg sivelestat drug preparation was administered once 24 hours before BCAS surgery; after successful modeling, 100 μL of 10 mg / kg sivelestat drug preparation was administered once a day, both by intraperitoneal injection, for 4 weeks.

[0070] It should be noted that the injections were stopped after 4 weeks (i.e., 28 days) in the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group. Although the administration was not continued on the 29th and 30th days after surgery, it did not affect the experimental test results.

[0071] 4. Experimental methods:

[0072] (2) Novel object recognition test: In order to evaluate non-spatial working memory, an object recognition test was performed. 28 days after surgery, mice were placed in a box of 30 cm × 30 cm × 30 cm, and two trials were conducted with an interval of 6 hours. In the first trial, two identical objects were placed in the box, and the mice were allowed to interact freely with the objects for 10 minutes. Interaction was recorded when the mouse's nose was <1 cm away from the object. In the second trial, either object was replaced with a new object, and the mouse explored the test area again for 10 minutes. The test result was the percentage of time spent exploring the new object to the total time spent exploring the old object and the new object.

[0073] (3) Y-maze test: 29 days after surgery, in order to evaluate the spatial working memory of mice, a Y-maze spontaneous change test was performed using a plastic Y-shaped runway. The Y-shaped runway has three identical arms (40 cm long, 12 cm high, and 3 cm wide), with a 120° angle between two adjacent arms. The arm where the mouse was initially placed was marked A, and the other two arms were marked B and C, respectively. The experiment allowed the mouse to freely explore the Y-maze for 10 minutes, and the total number and order of the mouse entering each arm were recorded with a video camera. Among them, the number of spontaneous alternations refers to the total number of times the mouse completed any of the six routes of ABC, ACB, BAC, BCA, CAB, and CBA; the total number of arm entries refers to the total number of times the mouse entered all individual arms of the Y-maze.

[0074] Spontaneous alternation rate (%) = number of spontaneous alternations / (total number of arm entries - 2) × 100%

[0075] Mice with fewer than 15 total arm entries during the test were excluded.

[0076] (4) Rotarod fatigue test: Mice were asked to run on a rotating rod starting at 5 rpm and accelerating to 40 rpm within 120 seconds, followed by a 5-minute maintenance period at 40 rpm. Four groups of mice were tested at 3 and 4 weeks after surgery. Each mouse was tested three times in a row, with a 15-minute interval. The time the mouse spent on the rotating rod was recorded. The mean of the three trials was calculated.

[0077] (5) Transmission electron microscopy was used to measure the myelin thickness of the corpus callosum. The specific operation was as follows: 30 days after surgery, the mice were deeply anesthetized and the brain tissue was perfused with 4% PFA containing 2.5% glutaraldehyde. The brain tissue was cut into 1 mm thick brain slices using a mouse coronal slice mold. On this basis, 1 mm thick slices of the left corpus callosum were extracted from 0.5 mm behind the anterior bregma. 3 As specimens. All specimens were post-fixed in osmium tetroxide, dehydrated through gradient ethanol concentrations, pre-embedded in propylene oxide, and flat-embedded in epoxy resin to obtain test specimens. The test specimens were observed by conventional transmission electron microscopy and examined under an electron microscope at 120 kV. Three to five images were collected at a magnification of 50,000 in randomly selected areas within the corpus callosum, and the inner and outer perimeters of the myelin sheaths of more than 50 axons per animal were analyzed using ImageJ software by researchers who were blinded to the experimental groups. The g-ratio is the ratio of the inner diameter to the outer diameter of the axon of each axon fiber.

[0078] (6) Luxol Fast Blue Stain (LFB): LFB staining is used to detect myelin changes in white matter areas. The commercial Luxol Fast Blue Stain (myelin stain) kit (ab150675, purchased from Abcam) was used according to the instructions to stain the myelin sheath in the corpus callosum region of mouse brain sections. TM White matter lesions in the corpus callosum were observed under a VS200 microscope and quantitatively analyzed using Image J software.

[0079] (7) Immunohistochemistry and image analysis: Prepare frozen sections of brain tissue and use the bleaching method to stain the corpus callosum of the mouse brain for MBP and olig2. Transfer 20 μm thick brain sections to a 24-well plate containing PBS buffer, rinse 3 times with PBS buffer, aspirate the liquid, add primary antibody, and incubate in a 4°C refrigerator for 16 hours. The brain sections incubated with the primary antibody were then washed 5 times with PBS buffer, and then incubated with a fluorescent-conjugated secondary antibody at room temperature for 1 hour, and then mounted on a slide with a mounting medium containing the nuclear staining reagent 4',6-diamidino-2-phenylindole (DAPI) (ab104139, purchased from Abcam). The primary and secondary antibodies used in this test method are shown in Table 1:

[0080] Table 1

[0081]

[0082] Images were collected using an Akoya Phenolmager HT fully automated quantitative pathology imager and analyzed using ImageJ software.

[0083] (8) Enzyme-linked immunosorbent assay (ELISA): A commercial ELISA kit (MELA20, purchased from R&D Systems) was used to measure the NE content in the mouse corpus callosum. The mouse corpus callosum tissue was minced and added to 50 μL of PBS. The brain tissue was ground using a sample grinder (Jing N9548 Intelligent Model, Beijing Hede Technology Co., Ltd.), and the cells were ultrasonically lysed for protein using an ultrasonic cell disruptor (JY92-IIN, Ningbo Xinzhi Biotechnology Co., Ltd.). The cells were centrifuged at 500 g for 15 minutes at 4°C. The supernatant sample was added to the ELISA kit and the NE content in the mouse corpus callosum was determined according to the manufacturer's instructions.

[0084] (9) NE activity assay: NE catalytic activity was determined using the synthetic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (M4765, purchased from Sigma). The reaction system for the NE enzyme activity assay consisted of 1 mmol / L substrate, 0.5 mol / L NaCl solution, and 0.1 mol / L Tris-HCl buffer (pH 8.0). Corpus callosum tissue homogenate samples were added to the NE enzyme activity assay reaction system and incubated at 37°C for 24 hours. After incubation, the amount of p-nitroaniline released (representing NE activity) was measured spectrophotometrically at 405 nm.

[0085] (10) Analysis of enzyme cleavage sites, N-terminal dimethyl labeling, and mass spectrometry: Recombinant human CNPase (aa 1-421, MBS1475160, purchased from MyBioSource, 4 μg) was mixed with human natural NE (SE563, purchased from Elastin products, 0.25 μg) and incubated at 37°C for 2 hours. The reaction was then terminated by adding SDS-PAGE denaturing protein loading buffer (B1012-5, purchased from Beijing Pulilai Gene Technology Co., Ltd.) and boiling to obtain the protease digestion product. The protease digestion product was sent to Beijing Biotech Biotechnology Co., Ltd. for dimethyl labeling and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The raw file of the mass spectrometry result was obtained. The human CNPase data (P09543) was matched in the UniProt database using the Byonic software to obtain the identification result.

[0086] (11) Tissue dissociation and cell separation: The brain tissue of newborn C57BL / 6 mice was weighed and enzymatically digested according to the instructions of the Mouse Neural Tissue Dissociation Kit (130-092-628, purchased from Miltenyi Biotec). The dissociated tissue was passed through a 100 μm cell strainer (BD Bioscience) and centrifuged at 300 g for 10 minutes at room temperature to obtain a cell pellet. The cell pellet was resuspended in D-PBS buffer (D8537, purchased from Sigma). Mouse oligodendrocyte precursor cells (OPCs) were enriched using the CD140α (PDGFRα) microbead kit (130-101-502, purchased from Miltenyi Biotec). The sorted OPCs were verified by immunofluorescence staining for cell-specific markers.

[0087] (12) Primary culture and treatment of oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes: Primary OPCs were obtained from C57BL / 6 mouse pups using a CD140a (PDGFRα) microbead kit (130-101-502, purchased from Miltenyi Biotec) according to the manufacturer's protocol. Primary OPCs were seeded onto poly-D-lysine / laminin-coated coverslips and expanded in growth medium for 3 days before the experiment. The growth medium was neurobasal medium (A2477501, purchased from Gibco) containing 2% B27 supplement (17504044, purchased from Gibco), 10 ng / mL PDGF-AA (100-13A, purchased from PeproTech), 10 ng / mL CNTF (450-50, purchased from PeproTech), and 1 ng / mL NT3 (450-03, purchased from PeproTech).

[0088] The OPCs obtained in the growth medium were differentiated for 6 days using differentiation medium, with half of the medium replaced every 2 days. The differentiation medium consisted of neurobasal medium containing 2% B27 supplement, 5 μg / mL insulin (I2643, purchased from Sigma-Aldrich), 40 ng / ml triiodothyronine (T3) (T6397, purchased from Sigma-Aldrich), and 10 ng / ml CNTF.

[0089] (13) Coomassie Brilliant Blue Staining: The protein product of NE (SE563, purchased from Elastin Products, 0.25 μg) digested with CNPase (aa 1-421, MBS1475160, purchased from MyBioSource, 4 μg) was subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue. The number and position of bands on the SDS-PAGE gel after Coomassie Brilliant Blue staining were used to determine the extent of NE digestion of CNPase.

[0090] (14) Cell immunofluorescence staining: OPC cells were inoculated into a 24-well plate with a slide one day in advance. The next day, staining was started when the OPC cells were about 70% fused. Specifically, the cells were fixed with 4% paraformaldehyde (PFA), then rinsed with PBS buffer 3 times, and after absorbing the liquid, the primary antibody was added and incubated in a 4°C refrigerator for 16 hours; then washed with PBS buffer 5 times, and the fluorescent-conjugated secondary antibody was added and incubated at room temperature for 1 hour. The slides were then sealed on a glass slide using a sealing agent. The primary and secondary antibodies used in this test method are shown in Table 2:

[0091] Table 2

[0092]

[0093] Images were captured using a Zeiss LSM 710 laser confocal microscope and analyzed using ImageJ software.

[0094] 5. Experimental results

[0095] 5.1NE expression is increased in the corpus callosum of patients and mice after chronic hypoperfusion

[0096] Regarding human specimens:

[0097] in, Figure 1 The NE in the corpus callosum of one of the human specimens in the control group and one of the patients with cerebral small vessel disease (CSVD) is shown in the embodiment of the disclosure. + The right side of the image of the CSVD group is the enlarged image in the white box, showing the NE + cell.

[0098] Figure 2 for Figure 1 NE in the corpus callosum of human specimens in the control group + Cells and Figure 1 NE in the corpus callosum of patients with midbrain small vessel disease (CSVD) + Quantitative plots of cells.

[0099] from Figure 1 and Figure 2 It can be seen that, regarding human specimens, the abundance of NE in the corpus callosum of patients with cerebral small vessel disease (CSVD) is significantly higher than that in human specimens from the control group.

[0100] Regarding experimental mice:

[0101] Figure 3 This example shows an enzyme-linked immunosorbent assay (ELISA) assay to assess NE protein concentration in the corpus callosum at different time points in sham-operated control mice and mice with CSVD white matter damage caused by bilateral common carotid artery stenosis (BCAS). Specifically, mice in the BCAS-induced CSVD white matter damage group did not receive any injections.

[0102] Figure 4 This is a graph showing the NE catalytic activity in corpus callosum homogenate samples from sham-operated control mice and BCAS-induced CSVD white matter damage mice at different time points using spectrophotometry in the embodiments of the present disclosure.

[0103] Depend on Figure 3and Figure 4 It can be seen that compared with the sham-operated mice, the NE protein concentration and NE catalytic activity in the corpus callosum of the BCAS-induced chronic hypoperfusion white matter injury group mice increased significantly 3 days after surgery and reached a peak 28 days after surgery.

[0104] 5.2NE induces oligodendrocyte precursor cell (OPC) and oligodendrocyte cell death in vitro

[0105] in, Figure 5 Representative optical microscopy images and quantitative graphs of active cells after oligodendrocyte precursor cells (OPCs) were treated with different concentrations of NE in the examples disclosed herein, wherein 0 nM represents treatment with only the solvent, which is a 0.9% NaCl solution.

[0106] Figure 6 Representative optical microscopy images and quantitative graphs of active cells after OPC cells were treated with 40 nmol / L NE at different time points in the examples of the present disclosure.

[0107] like Figure 5 and Figure 6 As shown, compared with the vehicle-treated group (ie, the control group), NE-treated oligodendrocyte precursors significantly reduced the number of OPC cells in a dose-dependent and time-dependent manner.

[0108] Figure 7 In the embodiment of the present disclosure, A2B5 positively labeled OPC cells were treated with solvent and NE for 5 hours, and caspase-3 + (CC3 + ) Representative confocal images and quantitative plots of cells. The solvent was 0.9% NaCl solution.

[0109] Figure 8 The graph is a quantitative graph of active cells in differentiated mature oligodendrocytes treated with different concentrations of NE according to the examples of the present disclosure.

[0110] Figure 9 In the embodiment of the present disclosure, CC3 in differentiated mature oligodendrocytes treated with solvent and NE were used. + Representative confocal images and quantitative plots of cells. The solvent was 0.9% NaCl solution.

[0111] Depend on Figure 8 It can be seen that compared with the solvent-treated group, the number of mature oligodendrocytes decreased in a concentration-dependent manner after NE treatment for 24 hours. Figure 7 and Figure 9 The immunostaining results showed that compared with the solvent-treated group, CC3 +The apoptotic cell count increased significantly, providing evidence that NE induces apoptosis in OPCs and mature oligodendrocytes.

[0112] 5.3NE's enzymatic cleavage effect on CNPase.

[0113] in, Figure 10 CNPase + Representative confocal images and quantitative graphs of cells in NG2-positive OPC cells.

[0114] Figure 11 Representative confocal images and mean fluorescence intensity quantification of CNPase staining of mature oligodendrocytes differentiated from the present invention after treatment with solvent and NE for 5 hours, respectively, wherein the solvent was 0.9% NaCl solution.

[0115] like Figure 10 and Figure 11 As shown in Figure 3, the CNPase protein levels in OPCs and mature oligodendrocytes were significantly decreased after NE treatment compared with the vehicle-treated group.

[0116] Figure 12 This is a diagram of the domain structure of the CNPase protein according to the disclosed embodiment.

[0117] from Figure 12 As shown in the figure, CNPase is composed of a polynucleotide kinase domain, a phosphodiesterase domain, and a C-terminal extension. In addition, isoform II contains an N-terminal mitochondrial targeting sequence (MTS, marked as M in the figure), which is removed after mitochondrial import; PNK refers to the polynucleotide kinase; 2H refers to the phosphodiesterase domain composed of two conserved histidine residues; and C refers to the C-terminal extension.

[0118] Figure 13 This is a diagram of the enzymatic cleavage products of recombinant human CNPase after being cleaved by human natural NE under different enzyme and substrate (i.e., NE and CNPase) mass ratios (right) and different enzymatic cleavage times (left) as evaluated by SDS-PAGE and Coomassie Brilliant Blue staining in the embodiments of the present disclosure.

[0119] Figure 14 This is a map of the NE cleavage site and sequence amino acid conservation in the human CNPase protein and mouse CNPase protein of the embodiments of the present disclosure.

[0120] from Figure 13 and Figure 14As can be seen in the figure, 11 primary cleavage sites were identified in human CNPase through N-terminal sequencing and mass spectrometry analysis. These sites are all located in the conserved 2H phosphodiesterase domain and are highly consistent with the NE enzyme cleavage site specificity recorded in the MEROPS database.

[0121] 5.4 Effects of sivelestat on histopathology, nerve fiber integrity, and neurological function.

[0122] in, Figure 15 These are representative images of the mouse corpus callosum stained with MBP, LFB, and Olig2, respectively, in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the disclosed embodiments.

[0123] Figure 16 30 days after surgery, the quantitative graph shows the mean fluorescence intensity of MBP in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group.

[0124] Figure 17 30 days after surgery, the figures show the extent of white matter lesions stained by LFB in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group in the examples of the present disclosure.

[0125] Figure 18 The expression of Olig2 in the corpus callosum of the mice in the sham operation group, the model group solvent administration control group, the model group low-dose administration group and the model group high-dose administration group 30 days after surgery in the disclosed embodiment was as follows: + Quantitative plots of cells.

[0126] from Figure 15-18 It can be seen that compared with the sham-operated control group, the MBP fluorescence intensity of the mice in the solvent-administered control group of the model group was significantly reduced, the integrity of the white matter was damaged, and the number of oligodendrocytes in the corpus callosum was reduced; while these symptoms were alleviated in the low-dose and high-dose groups of the model group, and the efficacy of the high-dose group was more obvious. This shows that treatment with sivelestat can indeed alleviate white matter lesions and oligodendrocyte reduction caused by cerebral hypoperfusion.

[0127] Figure 19 Representative electron micrographs of myelin sheaths in the corpus callosum of mice in the sham-operated control group, the model solvent-administered control group, the model low-dose administration group, and the model high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0128] from Figure 19It can be seen that the myelin sheath of the mice in the sham operation group remained intact with clear boundaries, while the myelin sheath of the mice in the solvent administration control group of the model group was severely damaged, with vacuoles and stratification (as shown by the red arrows).

[0129] Figure 20 This is a quantitative analysis graph of the g-ratio values ​​of nerve fibers (axons + myelin sheaths) in the corpus callosum of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0130] Figure 21 This is a scatter plot of the functional relationship between the g-ratio value and the axon diameter of the mice in the sham operation control group, the model group solvent administration control group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0131] Figure 22 This is a graph showing the axon diameters of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 30 days after surgery in the examples of the present disclosure.

[0132] like Figure 20-22 As shown in the figure, while there was no significant change in the axon diameter of all groups of mice, the g-ratio of the myelin sheath of the model group solvent-administered control group mice increased compared with the sham-operated control group, indicating that the BCAS disease model leads to a decrease in the thickness of the myelin sheath. Compared with the model group solvent-administered control group, the g-ratio of the myelin sheath of BCAS mice treated with sivelestat was significantly reduced, and the g-ratio of the high-dose model group was closer to the sham-operated group. Figure 19 It can also be clearly seen that the myelin integrity of mice in the high-dose administration group of the model group was better, indicating that inhibiting NE with sivelestat can indeed reduce the myelin damage of nerve fibers after cerebral hypoperfusion.

[0133] Figure 23 Graphs showing the results of the rotarod fatigue test of mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group at different time points in the disclosed embodiments.

[0134] like Figure 23 As shown in the results, compared with the sham-operated control group, the time that the model group's vehicle-administered control group mice stayed on the rotating rod in the rotarod fatigue test was significantly reduced. Compared with the model group's vehicle-administered control group, mice treated with sivelestat showed dose-dependent improvements in motor balance function, with statistically significant improvements occurring at a high dose of 10 mg / kg.

[0135] Figure 2428 days after surgery, the results of the object recognition test were performed on mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group in the examples of the present disclosure.

[0136] like Figure 24 As shown, in the novel object recognition test, the exploratory preference for novel objects in the vehicle-administered control group of the model group was significantly reduced after training compared with the sham-operated control group mice. In mice treated with sivelestat, this reduction was improved in a dose-dependent manner.

[0137] Figure 25 This is a graph showing the results of a Y-maze test on mice in the sham operation control group, the model group solvent administration control group, the model group low-dose administration group, and the model group high-dose administration group 29 days after surgery in the examples of the present disclosure.

[0138] like Figure 25 As shown in the Y-maze test, 10 mg / kg sivelestat treatment significantly restored the short-term spatial working memory function of BCAS mice, indicating that sivelestat can indeed partially reverse the effects of cerebral hypoperfusion on neurological function.

[0139] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0140] The foregoing description is intended only to provide specific embodiments of the present disclosure, intended to enable those skilled in the art to understand and implement the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is not intended to be limited to the embodiments described herein, but rather to be construed in the broadest manner consistent with the principles and novel features disclosed herein.

Claims

1. Use of sivelestat in the preparation of a drug for treating white matter damage caused by chronic global cerebral hypoperfusion.

2. The use according to claim 1, characterized in that The dosage form of the drug is lyophilized powder or injection.

3. The use according to claim 1, characterized in that The medicine also includes one or more pharmaceutically acceptable carriers or excipients.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives.

Images