Use of kallikrein 1 or a derivative thereof in the treatment of VCI, PSCI or CSVD

Abstract

Use of kallikrein 1 or a derivative thereof in the treatment of vascular cognitive impairment (VCI), post-stroke cognitive impairment (PSCI) or cerebral small vessel disease (CSVD).

Description

[Technical field]

[0001] The present invention relates to the use of kallikrein 1 or a derivative thereof in the treatment of vascular cognitive impairment (VCI), post-stroke cognitive impairment (PSCI) or cerebral small vessel disease (CSVD). [Background technology]

[0002] With the development of society and the aging of the population, the incidence of cerebrovascular disease is increasing year by year, which has a serious impact on the quality of life of patients and their families. In 1993, Hachinski and Bowler first proposed the concept of vascular cognitive impairment (VCI). This is a condition that is characterized by the risk factors for cerebrovascular disease (e.g., hypertension, diabetes, hyperlipidemia, etc.), obvious cerebrovascular disease (e.g., cerebral infarction and cerebral hemorrhage, etc.), or obvious It is a series of syndromes ranging from mild cognitive impairment to dementia caused by minor cerebrovascular diseases (e.g. white matter sparing and chronic cerebral ischemia) (2019 China Guidelines for the Treatment of Vascular Cognitive Disorder, China Guidance Guidelines for the Treatment of Vascular Cognitive Disorder in China's Guidance Guidelines for the Prevention and Treatment of Stroke (2021 Edition)).

[0003] Patients with vascular cognitive impairment (VCI) have cognitive impairment and psychiatric and behavioral symptoms. In some patients, the cognitive impairment is mainly functional impairment in abstract thinking, concept formation and transformation, flexibility of thought, and information processing speed, and memory is preserved. In other patients, impairment is seen in multiple domains, and memory is also significantly impaired. Medications for the treatment of cognitive impairment in VCI include cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists. Mental and behavioral symptoms of VCI include abnormalities in perception, thinking, mood, and behavior, and lethargy, depression, agitation, and aggression are common. Symptomatic medications such as selective serotonin reuptake inhibitors, which are antidepressants, are available for treatment (China's Guidelines for the Treatment of Vascular Cognitive Impairment). The main subtypes of VCI include post-stroke cognitive impairment (PSCI), cognitive impairment due to cerebral small vessel disease (CSVD), and Alzheimer's disease (AD) with vascular lesions.

[0004] Post-stroke cognitive impairment emphasizes the cognitive impairment caused by stroke, and similarly shows cognitive damage and mental and behavioral symptoms. Considering that PSCI, VCI, and AD have some commonality in neuropathological and neurobiochemical mechanisms, China's "Expert Consensus on the Management of Post-Stroke Cognitive Impairment 2021" recommends that in treating PSCI, the research and evidence of VCI, AD, etc. should be taken into consideration, and that acetylcholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists (memantine) should generally be used for its cognitive impairment, and antidepressants should generally be used for post-stroke depression. According to the "2021 ESO / EAN Joint Guideline: Post-Stroke Cognitive Impairment" recently published by the European Stroke Organization, there is still no treatment for post-stroke cognitive impairment. Among the trials that this guideline focuses on, one focused on post-stroke cognitive impairment, but no positive results were obtained. The guideline also points out that there is a significant lack of high-quality data from randomized controlled trials for PSCI.

[0005] Cerebral small vessel disease (CSVD) is a group of clinical, imaging, and pathological syndromes caused by multiple risk factors affecting small arteries, arterioles, capillaries, venules, and venules in the brain, and is classified into acute CSVD and chronic CSVD. Acute CSVD can lead to ischemic stroke. It is currently recommended that patients with chronic CSVD be treated with reference to the prevention and treatment of acute ischemic stroke (e.g., blood pressure reduction, thrombolysis, antiplatelet, lipid reduction, etc.). Patients with chronic CSVD show symptoms such as cognitive impairment, motor disorder, emotional disorder, and bowel disorder, and after a clear diagnosis, symptomatic treatment is generally recommended. For example, cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists are used for cognitive impairment due to CSVD, and antidepressants such as serotonin reuptake inhibitors are generally used for depression ("Chinese Cerebral Small Vessel Disease Medical Experts' Common Understanding"). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] "2019 Chinese Guidelines for the Treatment of Vascular Cognitive Disorders" [Non-Patent Document 2] "China's Guiding Guidelines for Stroke Prevention and Treatment (2021 Edition)" [Non-Patent Document 3] "Professional consensus on managing post-stroke cognitive impairment 2021" [Non-Patent Document 4] 2021 ESO / EAN Joint Guideline: Post-Stroke Cognitive Impairment [Non-Patent Document 5] "Common understanding of Chinese cerebral small vessel disease specialists" Summary of the Invention [Problem to be solved by the invention]

[0007] The first technical problem to be solved by the present invention is to provide a new drug for treating vascular cognitive impairment. The second technical problem to be solved by the present invention is to provide a new drug for treating cerebral small vessel disease. [Means for solving the problem]

[0008] The first technical problem to be solved by the present invention is to provide a new drug for treating vascular cognitive dysfunction, specifically, to provide the use of kallikrein 1 or a derivative thereof in the manufacture of a drug for treating or ameliorating vascular cognitive dysfunction.

[0009] The present invention also provides a pharmaceutical composition for treating or ameliorating vascular cognitive dysfunction, the composition comprising kallikrein 1 or a derivative thereof.

[0010] The present invention also provides a method of treating or ameliorating vascular cognitive impairment, comprising administering to a patient kallikrein 1 or a derivative thereof.

[0011] The vascular cognitive dysfunction includes, but is not limited to, post-stroke cognitive dysfunction, cognitive dysfunction due to cerebral small vessel disease, Alzheimer's disease accompanied by vascular lesions, and the like.

[0012] Preferably, said vascular cognitive dysfunction is post-stroke cognitive dysfunction. Preferably, the vascular cognitive impairment is cognitive impairment due to cerebral small vessel disease. Preferably, the vascular cognitive dysfunction is Alzheimer's disease accompanied by vascular lesions.

[0013] The second technical problem to be solved by the present invention is to provide a new drug for treating cerebral small vessel disease, specifically, to provide the use of kallikrein 1 or a derivative thereof in the manufacture of a drug for treating or ameliorating cerebral small vessel disease.

[0014] The present invention also provides a pharmaceutical composition for treating or ameliorating cerebral small vessel disease, the composition comprising kallikrein 1 or a derivative thereof.

[0015] The present invention also provides a method for treating or ameliorating cerebral small vessel disease, comprising administering kallikrein 1 or a derivative thereof to a patient.

[0016] The kallikrein 1 may be kallikrein 1 of a natural extract or recombinant kallikrein 1. The kallikrein 1 derivative may be a full-length protein of kallikrein 1, or a partial protein, mutant, fusion protein, or various types of modified forms thereof.

[0017] Preferably, the kallikrein 1 derivative is kallikrein 1 modified with polyethylene glycol.

[0018] The kallikrein 1 or its derivative may be administered alone or in combination with other drugs for treating or ameliorating cerebral small vessel disease or vascular cognitive dysfunction (e.g., cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists, etc.).

[0019] The administration method includes, but is not limited to, injection, oral administration, etc. The injection method includes, but is not limited to, intravenous injection, subcutaneous injection, intramuscular injection, etc.

[0020] Widely accepted animal models of vascular cognitive impairment include rat models using 4-vessel occlusion (4-VO), modified 4-VO, three-stage 4-VO, 2-vessel occlusion (2-VO), modified 2-VO, and modified common carotid artery occlusion (mCCAO), as well as mouse models using bilateral CCA stenosis (BCAS) and asymmetric CCA surgery (ACAS) (Research Progress in Animal Models of Vascular Cognitive Impairment).

[0021] Widely accepted animal models of cerebral small vessel disease mainly include the bilateral common carotid artery ligation model, the bilateral common carotid artery stenosis model (BCAS), and the stroke-prone spontaneously hypertensive rat model (Chinese Expert Consensus on Translational Research of Cerebral Small Vessel Disease).

[0022] The BCAS model, a well-accepted experimental animal model for the study of vascular cognitive impairment and cerebral small vessel disease, shows that animals' cerebral blood flow is significantly reduced, which leads to chronic hypoperfusion injury, increased blood-brain barrier permeability, white matter damage, and further memory dysfunction, motor function impairment, etc.

[0023] The research results of the present invention show that administration of kallikrein 1 or its derivatives to BCAS model mice significantly improved the animals' impaired coordinated motor function, impaired muscle strength of the forelimbs, working memory, and spatial learning and memory functions. Therefore, kallikrein 1 or its derivatives can be used to treat vascular cognitive dysfunction and cerebral small vessel disease.

[0024] In addition, post-stroke cognitive impairment, cognitive impairment due to cerebral small vessel disease, and Alzheimer's disease with vascular lesions are all important subtypes of vascular cognitive impairment, and the clinical treatment of each uses similar symptomatic drugs. For example, for cognitive impairment, treatment guidelines and expert consensus for vascular cognitive impairment, post-stroke cognitive impairment, and cerebral small vessel disease generally recommend the use of acetylcholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists (memantine), and drug treatment for mental and behavioral symptoms such as depression generally recommends the use of antidepressants. Therefore, kallikrein 1 or its derivatives can be used to treat various subtypes of vascular cognitive impairment, such as post-stroke cognitive impairment, cognitive impairment due to cerebral small vessel disease, and Alzheimer's disease with vascular lesions.

[0025] The kallikrein-kinin system (KKS) is involved in various physiological and pathological processes, including the regulation of cardiovascular, renal, and nervous system functions. The KKS system includes kallikrein, kininogen, kinin, kinin receptors (B1 and B2 receptors), and kininase. Kallikrein, also called kininogenase or kallidinogenase, is a type of serine protease and is divided into two types, plasma kallikrein 1 (PK) and tissue kallikrein 1 (TK), both of which exert very important physiological functions. Currently, human tissue kallikrein is thought to consist of at least 15 members (KLK1-KLK15), of which tissue kallikrein 1 (KLK1) has been studied more extensively. KLK1 converts kininogen into kinin and acts on the corresponding receptor to exert a series of biological functions. Two types of injectable kallikrein 1 are available in China, with indications including microcirculatory disorders and mild to moderate acute ischemic stroke.

[0026] Vascular cognitive impairment and cerebral small vessel disease are quite different from the approved indications of KLK1. In terms of pathological mechanism, acute ischemic stroke is caused by vascular wall lesions due to various causes, which cause stenosis or occlusion in the lumen of the main or branch arteries of the cerebral artery, or the formation of a thrombus, resulting in a decrease in local cerebral blood flow or the interruption of blood supply, which causes ischemic or hypoxic necrosis in the brain tissue, resulting in local nervous system symptoms and signs. On the other hand, vascular cognitive impairment is a series of syndromes ranging from mild cognitive impairment to dementia due to risk factors for cerebrovascular disease (e.g., hypertension, diabetes, hyperlipidemia, etc.), obvious cerebrovascular disease (e.g., cerebral infarction, cerebral hemorrhage, etc.), or unclear cerebrovascular disease (e.g., white matter sparing, chronic cerebral ischemia).

[0027] In the pathogenesis of cerebral small vessel disease, dysfunction of the neurovascular unit (NVU) plays an important role, and CSVD can occur when the NVU undergoes structural or functional changes for some reason. Commonly considered mechanisms of pathogenesis include chronic cerebral ischemia and hypoperfusion, endothelial dysfunction, destruction of the blood-brain barrier, impaired interstitial fluid perfusion, inflammatory responses, and genetic factors. Different mechanisms mutually influence each other (the neurovascular unit coming of age:a journey through neurovaseular coupling in health and disease).

[0028] In addition, the diagnosis and treatment of acute ischemic stroke are very different from those of vascular cognitive impairment and cerebral small vessel disease. The most effective treatment for acute ischemic stroke is to perform endovascular recanalization therapy (intravenous thrombolysis, mechanical thrombectomy, angioplasty, etc.) within a time window, and the success rate of treatment is closely related to the onset time. Drug treatment includes the use of antiplatelet, anticoagulant, fibrolytic, volume expanding, vasodilator, statin drugs, neuroprotective drugs, etc. VCI and PSCI often present with cognitive dysfunction and psychiatric and behavioral symptoms. Drugs for cognitive dysfunction in VCI and PSCI include cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists, and symptomatic drugs for psychiatric and behavioral symptoms include selective serotonin reuptake inhibitors, which are antidepressants. Patients with chronic cerebral small vessel disease may experience symptoms such as cognitive impairment, motor disorders, emotional disorders, and bowel disorders, and symptomatic treatment is generally recommended after a clear diagnosis. For example, cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists are used to treat cognitive impairment due to cerebral small vessel disease, and antidepressants such as serotonin reuptake inhibitors are commonly used to treat depression.

[0029] Through research, the present invention has revealed that kallikrein 1 or its derivatives can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, cerebral small vessel disease, etc. It provides a new method for treating vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease, which is different from the approved indications of kallikrein 1. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows that KLK1 and its mutants activate phosphorylation of the downstream CREB protein. [Diagram 2] FIG. 2 shows that Lys-BK activates the phosphorylation of downstream ERK1 / 2 and CREB proteins. [Diagram 3] FIG. 3 shows the effect of drugs on the latency to fall in the Rotarod test on the 13th day in Experiment 1. [Figure 4] FIG. 4 shows the effect of drugs on the time to fall in the Rotarod test on the 26th day after administration in Experiment 1. [Diagram 5] FIG. 5 shows the effect of drugs on the latency to fall in the wire hang test on the 20th day in Experiment 1. [Figure 6] FIG. 6 shows the effect of drugs on the latency to fall in the wire hang test on the 28th day in Experiment 1. [Figure 7] FIG. 7 shows the effects of drugs on the alternation score in the Y-maze test on the 29th day in Experiment 1. [Figure 8] FIG. 8 shows the effects of drugs on the alternation score in the Y-maze test on day 44 in Experiment 1. [Figure 9] FIG. 9 shows the effects of drugs on the latency to reach the platform in the Morris water maze test on the 48th day in Experiment 1. [Figure 10] FIG. 10 shows the effect of drugs on the latency to fall in the Rotarod test in Experiment 2. [Figure 11] FIG. 11 shows the effect of drugs on the latency to fall in the wire hang test in experiment 2. [Figure 12] FIG. 12 shows the effects of drugs on the latency to reach the platform (movement distance in the third quadrant) in the Morris water maze test in Experiment 2. [Figure 13] FIG. 13 shows the effects of drugs on the latency to reach the platform (behavioral time in the third quadrant) in the Morris water maze test in Experiment 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Unless otherwise specified, technical terms or abbreviations in the present invention have the following meanings: hKLK1: refers to unmutated human tissue kallikrein 1 whose sequence is identical to native human tissue kallikrein 1, including various homologues of human KLK1, including but not limited to KLK1 registered in Genbank accession numbers AAA59455.1, NP002248.1, AAA36136.1, AAP35917, AAU12569, etc. In a specific embodiment, the sequence is as set forth in SEQ ID NO:5.

[0032] There are three glycosylation sites in KLK1 (N78, N84, and N141): they refer to asparagine at positions 78, 84, and 141 of the KLK1 amino acid sequence, respectively, and the corresponding N-glycosylation triad sequence motifs are NMS, NHT, and NFS, respectively. N stands for asparagine, M stands for methionine, S stands for serine, H stands for histidine, T stands for threonine, and F stands for phenylalanine.

[0033] Hyperglycosylated hKLK1: non-mutated hKLK1 that is hyperglycosylated, i.e., the glycosylation sites N78, N84, and N141 all have numerous glycosylation modifications.

[0034] Hypoglycosylated hKLK1: non-mutated hKLK1 with hypoglycosylation. That is, there are many glycosylation modifications at both glycosylation sites N78 and N84, and there is no glycosylation modification or no glycosylation modification at glycosylation site N141. The recombinant hyperglycosylated hKLK1 and hypoglycosylated hKLK1 can be isolated by conventional purification methods, such as hydrophobic chromatography, anion-linked chromatography, cation-linked chromatography, or a combination thereof. The applicant of the present invention unexpectedly discovered that the activity of hypoglycosylated KLK1 is much higher than that of hyperglycosylated KLK1.

[0035] PEG-hKLK1 (hyperglycosylated): hyperglycosylated hKLK1 modified with polyethylene glycol. The hyperglycosylated hKLK1 refers to hKLK1 having the above-mentioned non-mutated hyperglycosylation modification.

[0036] PEG-hKLK1 (hypoglycosylated): Hypoglycosylated hKLK1 modified with polyethylene glycol. The hypoglycosylated hKLK1 refers to hKLK1 having the above-mentioned non-mutated hypoglycosylation modification.

[0037] hKLK1X: a mutant of hKLK1. KLK1 is an enzyme with many glycosylation modifications. N-glycosylation is the most prominent type of glycosylation modification of glycoprotein drugs, in which sugar chains are attached to specific asparagine in the nascent peptide chain through the free radical -NH2. The triad sequence motif of the N-glycosylation site must be NXS or NXT, where N represents asparagine, S represents serine, T represents threonine, and X represents an amino acid other than proline. If the above conditions are not met, asparagine will not undergo N-glycosylation. hKLK1X1, hKLK1X2, hKLK1X3, and hKLK1X4 represent different mutants, and their sequences are as shown in SEQ ID NO:1, 2, 3, and 4, respectively. These mutants were obtained by site-directed mutagenesis of the glycosylation site N141, where asparagine was mutated to four different amino acids, namely, the polar neutral amino acid glutamine (Gln), the acidic amino acid aspartic acid (Asp), the basic amino acid arginine (Arg), and the aliphatic amino acid alanine (Ala). The resulting hKLK1 mutants did not contain the N-glycosylation motif NFS and contained only two glycosylation sites, which resulted in more homogeneous products and higher yields of hypoglycosylated hKLK1. Unexpectedly, we also found that the activity of the mutated hypoglycosylated KLK1 was much higher than that of the unmutated hypoglycosylated KLK1.

[0038] The above-mentioned mutants are for illustrative purposes only and do not limit the scope of the present invention. The following methods all modify the original NFS sequence to avoid forming an N-glycosylation motif, so that N141 is not glycosylated, and therefore the product is more uniform and the yield of low-glycosylated hKLK1 is higher. For example, the amino acid N (i.e., N141) in NFS is mutated, and 0, 1, or 2 amino acids in F and S are mutated to any other amino acid; the F (i.e., F142) in NFS is mutated to proline, and 0, 1, or 2 amino acids in N and S are mutated to any other amino acid; or the S (i.e., S143) in NFS is mutated to any amino acid except threonine, and 0, 1, or 2 amino acids in N and F are mutated to any other amino acid. Unexpectedly, they discovered that the activity of the mutated underglycosylated KLK1 was much higher than that of the non-mutated underglycosylated KLK1.

[0039] PEG-hKLK1X: hKLK1 mutant modified with polyethylene glycol.

[0040] KLK1 derivatives: Contains the full-length KLK1 protein and also contains partial KLK1 proteins. The present invention also includes the KLK1 mutants of the present invention, as well as proteins obtained by further mutation based on the KLK1 mutants of the present invention, fusion proteins (including, but not limited to, albumin fusion, Fc fusion, etc.), and various types of modifications (including, but not limited to, polyethylene glycol modified proteins).

[0041] Polyethylene glycol: abbreviated as PEG, is usually formed by polymerization of ethylene oxide and is available in branched, linear and multi-armed forms. Generally, those with a molecular weight of 20,000 or less are called PEG, and those with a higher molecular weight are called PEO. Ordinary polyethylene glycol has one hydroxyl group at each end, and blocking one end with a methyl group results in methoxypolyethylene glycol (mPEG).

[0042] Polyethylene glycol modifier: abbreviated as PEG modifier, refers to a polyethylene glycol derivative with functional groups, which is an activated polyethylene glycol used to modify protein and polypeptide drugs. The polyethylene glycol modifier used in the present invention is purchased from Jiangsu Zhonghong Bioengineering Drug Discovery Research Institute Co., Ltd. or Beijing Keykai Technology Co., Ltd. The actual molecular weight of a PEG modifier with a specific molecular weight may be 90%-110% of the listed value. For example, the molecular weight of PEG5K may be 4.5kDa-5.5kDa.

[0043] PEG5K used in the examples specifically refers to M-SPA-5K, which is a linear methoxy polyethylene glycol succinimidyl propionate having a molecular weight of about 5 kDa and a structural formula as shown in formula (1), where n is an integer of 105 to 110.

[0044] [ka]

[0045] PEG10K used in the examples specifically refers to M-SPA-10K, which is a linear methoxy polyethylene glycol succinimidyl propionate having a molecular weight of about 10 kDa and has the structural formula shown in formula (1), where n is an integer of 220 to 225.

[0046] PEG30K used in the examples specifically refers to Y-PALD-30K, which is a branched polyethylene glycol propionaldehyde having a molecular weight of about 30 kDa and has the structural formula shown in formula (2), where m is an integer of 335 to 340.

[0047] [ka]

[0048] PEG40K used in the examples specifically refers to Y-PALD-40K, which is a branched polyethylene glycol propionaldehyde having a molecular weight of about 40 kDa and has the structural formula shown in formula (2), where m is an integer of 450 to 455.

[0049] The polyethylene glycol modifying agent used for modifying KLK1 is not limited to the above-mentioned polyethylene glycol modifying agents used in the specific examples, and other known polyethylene glycol modifying agents may be used.

[0050] The polyethylene glycol modified KLK1 used in the following examples is a polyethylene glycol modified protein obtained by polyethylene glycol modification and purification using a method known in the art. For example, the polyethylene glycol modified KLK1 can be prepared by referring to Chinese Patent CN109498815A, Chinese Patent CN107760661A or prior invention CN202111353294.2. EXAMPLES

[0051] Example 1 In vitro activity measurement of recombinant human kallikrein 1 (hKLK1) and its mutant (hKLK1X) 1. In vitro activity evaluation using artificial substrates KLK1 exerts its biological function in vivo by catalyzing the hydrolysis of low molecular weight kininogen LMWK to release lysylbradykinin. This hydrolysis reaction involves the cleavage of a peptide bond at the carboxyl end of arginine (Arg). Therefore, it is possible to evaluate the in vitro biological activity of recombinant hKLK1 and its mutants through the detection of the production of paranitroaniline (PNA) at 405 nm based on the hydrolysis of the amide bond between Arg and paranitroaniline in the chromogenic artificial synthetic substrate S-2266 (HD-Val-Leu-Arg-PNA). The activity unit IU is defined as the amount of enzyme that hydrolyzes 1 μmol of S-2266 to produce PNA in one minute under the conditions of 37°C and pH 8.0. The reaction system consists of 200 μl of 20 mM trishydroxymethylaminomethane buffer, 10 μl of the test sample, and 20 μl of 20 mM S-2266 substrate solution. The reaction system was placed in a water bath at 37°C for exactly 10 minutes, and 20 μl of 50% acetic acid solution was added to stop the reaction. The amount of PNA produced in the reaction system was quantified by a standard curve fitted based on different concentrations of PNA standards. Using the above method, the in vitro biological activities of hyperglycosylated and hypoglycosylated hKLK1 and hKLK1 mutants were measured.

[0052] The results are shown in Table 1. The activities of the mutant (hKLK1X1, hKLK1X2, hKLK1X3, hKLK1X4) samples were all higher than the non-mutated underglycosylated hKLK1 sample.

[0053] [Table 1]

[0054] 2. In vitro activity measurement using natural substrates 1. Enzyme reaction and liquid phase measurement KLK1 exerts its biological function by catalyzing the hydrolysis of low molecular weight kininogen LMWK in vivo, liberating lysylbradykinin. In this example, the substrate (low molecular weight kininogen LMWK) and enzyme (KLK1 or its PEG-modified form) are reacted at different ratios to compare the production of the effector molecule bradykinin. The effector molecules produced are separated by reversed-phase chromatography, the peak area of ​​the product is calculated, and the bradykinin production curves are drawn when the substrate and enzyme react at different ratios, and the amount of effector molecules produced by different test samples under the same reaction conditions is compared. In this way, the in vivo action effects of different test sample samples are indirectly compared by simulating the action mode in the body in vitro.

[0055] The Kb in the test sample is KLK1 extracted from porcine pancreas, and PEG-Kb is Kb modified with PEG10K. PEG10K-hKLK1X1 and PEG-Kb were prepared by the usual method of drug modification with polyethylene glycol.

[0056] The samples were mixed and reacted according to Table 2 (in the case of PEG-modified samples, the molar mass of the active substance to be modified was used as the conversion factor). The mixed samples were placed in a 37°C incubator and incubated for 15 minutes while being accurately timed, and the reaction was stopped by adding 50% acetic acid solution at a volume ratio of 10:1.

[0057] [Table 2]

[0058] After the reaction was stopped, the samples were placed in a tabletop centrifuge and centrifuged at 12000 rpm for 5 minutes to obtain the supernatant. The measurements were performed using a Waters ACQUITY UPLC H-Class. Mobile phase A was 0.1% TFA-H2O, mobile phase B was 0.1% TFA-ACN, the measurement wavelength was 214 nm, the column temperature was 30°C, the sample loading volume was all 10 μl, the flow rate was 0.2 mL / min, the run time was 35 minutes, and the run gradient was as follows:

[0059] [Table 3]

[0060] 2, results Based on the results of the UPLC chromatogram of the enzyme reaction mixture, the peak with a retention time of 13±0.5 min (peak position of bradykinin) in the chromatogram of the reaction mixture was integrated to obtain the sum, and the concentration of bradykinin produced in the enzyme reaction was calculated by comparing with the peak area of ​​bradykinin per unit mass (1 mg / mL, 10 μL sample loading amount). The amount of bradykinin produced was calculated based on the total reaction system in Table 2, and finally converted to the amount of bradykinin produced per 1 mg enzyme (μg / mg) under each molar ratio condition. The results are shown in Table 4.

[0061] [Table 4]

[0062] As can be seen from the results of the measurements based on the natural substrate, the overall trend was that the amount of bradykinin produced by the enzymatic reaction of the modified protein was lower than that of the unmodified sample. This is in line with the properties of PEG-modified proteins. Compared with the original protein, the PEG-modified protein maintains a more gentle enzymatic reaction, so it can produce effector molecules continuously. In addition, the effect of KLK1 drugs is based on the regulation of the kallikrein-kinin system (KKS) in the body, which includes the production and elimination of effector molecules. Obviously, the gentle and sustained enzymatic reaction process effectively reduces the elimination mechanism in the KKS, so the PEG-modified KLK1 can exert its biological effect more stably and effectively.

[0063] Example 2 Activation effect of KLK1 on downstream signaling of B2 receptor CHO-K1 cells in the logarithmic growth phase (containing the B2 receptor) were harvested and the number of viable cells was counted using a hemocytometer. The viable cell suspension was diluted to 3 × 106 The final cell concentration was adjusted to 3 × 10 cells / mL. 5 The cells were seeded in a 6-well cell culture plate so that there were 10 cells / well. The cells were cultured in a CO2 incubator at 37.0 °C and 5.0% CO2 for 24 to 48 hours. When the cell density in each well grew to 90% or more, the cells were washed twice with PBS. Samples were prepared in advance using F12 medium (KLK1 and LMWK were mixed in a mass ratio of 1:10, and the reaction system was F12 medium and reacted for 15 minutes). After the reaction was completed, 1 ml of the sample was added to each well, and the final concentration of KLK1 was 0.1 μM, and F12 medium was used as the control group. After the addition was completed, the cell culture plate was cultured in a 37.0 °C, 5.0% CO2 incubator for 0.5 hours. After the culture was completed, the 6-well plate was removed from the incubator, washed twice with PBS, and finally the PBS was completely removed by suction. The prepared protein lysis solution (RIPA, PMSF, phosphatase inhibitor) was added at 80 μL / well, and the bottom of the well was scraped back and forth with a scraper to accelerate cell lysis. The scraping time was 1 min. The cell debris and lysis solution were transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein supernatant was carefully aspirated and mixed with 5 × Loading Buffer in a 4:1 volume ratio, and then boiled in a metal bath at 100 °C for 10 min. After cooling the samples at room temperature, they were stored at 4 °C for short-term storage and at -20 °C for long-term storage. The protein samples and pre-stained markers were loaded in the same amount onto 10% SDS-PAGE, and operations such as gel running, membrane transfer, blocking, antibody incubation, and luminescence identification were performed.

[0064] Cyclic adenosine monophosphate effector binding protein ( C AMP-response element binding protein (CREB) is a signaling molecule that acts directly downstream after B2R (B2 receptor) activation. CREB activation can prevent the occurrence of post-ischemic inflammatory responses and neuronal damage, so the expression level of CREB reflects to a certain extent the ability of the test substance to activate B2R, and also indirectly reflects the effect of the neuroprotective effect it exerts.

[0065] Based on the B2-NFAT-CHO-K1 cell line, we demonstrated that KLK1 catalyzes the generation of active molecules from low molecular weight kininogen LMWK, and the generated active molecules bind to the B2 receptor to activate the downstream signal CREB protein. As shown in Figure 1, KLK1 can activate the phosphorylation of the downstream CREB protein, which is consistent with the mechanism of action of the drug. Through the determination of the phosphorylation level of the downstream CREB protein, the in vitro biological activity of the samples was qualitatively evaluated. The results showed that hKLK1X1, hKLK1X2, hKLK1X3, hKLK1X4, low-glycosylated hKLK1, and high-glycosylated hKLK1 all showed the activity of activating the downstream pathway by reacting with LMWK.

[0066] Example 3: Lysylbradykinin (Lys-BK) activates downstream signals of B2 receptor CHO-K1 (B2 receptor-containing) cells in the logarithmic growth phase were harvested and the number of viable cells was counted using a hemocytometer. The viable cell suspension was diluted to 3 × 10 6 The final cell concentration was adjusted to 3 × 10 cells / mL. 5 The cells were seeded in a 6-well cell culture plate at 100 cells / well. The cells were cultured in a CO2 incubator at 37.0°C and 5.0% CO2 for 24 to 48 hours. When the cell density in each well reached 90% or more, the cells were washed twice with PBS. A sample (Lys-BK) was prepared in advance using F12 medium, and 1 ml of the sample was added to each well. The final concentration of Lys-BK was 1 μM, and F12 medium was used as the control group. After addition, the cell culture plate was cultured in an incubator at 37.0°C and 5.0% CO2 for 0.5 hours. After culture, the 6-well plate was removed from the incubator and The wells were washed twice with PBS, and finally the PBS was completely aspirated off. The prepared protein lysis solution (RIPA, PMSF, phosphatase inhibitor) was added at 80 μL / well, and the bottom of the well was scraped back and forth with a scraper to accelerate cell lysis. The polishing time was 1 min. The cell debris and lysis solution were transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein supernatant was carefully aspirated and mixed with 5 × Loading Buffer in a 4:1 volume ratio, and then boiled in a metal bath at 100 °C for 10 min. After cooling the samples at room temperature, they were stored at 4 °C for short-term storage and at -20 °C for long-term storage. The protein samples and pre-stained markers were loaded in the same amount onto 10% SDS-PAGE, and operations such as gel running, transfer to membrane, blocking, antibody incubation, and luminescence identification were performed.

[0067] Extracellular signal-regulated protein kinases 1 / 2 (ERK1 / 2), cyclic adenosine monophosphate effector binding protein ( C AMP-response element binding protein (CREB) is a signaling molecule that acts directly downstream after B2R activation. Activation of CREB and ERK1 / 2 can prevent the occurrence of postischemic inflammatory responses and neuronal damage, so their expression levels reflect to some extent the ability of the test substance to activate B2R, and also indirectly reflect the neuroprotective effect it exerts.

[0068] Based on the B2-NFAT-CHO-K1 cell line, we demonstrated that lysylbradykinin Lys-BK binds to the B2 receptor and activates the downstream signals CREB and p-ERK1 / 2 proteins. As shown in Figure 2, Lys-BK can activate the phosphorylation of downstream CREB and p-ERK1 / 2 proteins.

[0069] Example 4: Efficacy of KLK1-PEG in BCAS mouse model Experiment 1 I. Group assignment and experimental design A bilateral common carotid artery spring constriction procedure was performed to create a mouse model of cerebral small vessel ischemic injury caused by bilateral common carotid artery stenosis (BCAS).

[0070] Preparation of BCAS cerebral small vessel injury model: The BCAS cerebral small vessel ischemic injury model was prepared to subject the forebrain of mice to persistent chronic ischemia. Mice were anesthetized with isoflurane. First, the mouse was placed in the induction anesthesia box of the anesthesia machine to induce anesthesia. The mouse was then fixed on its back and connected to a breathing mask, the skin was shaved and disinfected, the midline of the neck was incised, and both common carotid arteries were isolated. A custom-made spring (spring material: imported piano wire, dimensions: wire diameter 0.08 mm, inner diameter 0.18 mm, pitch: approximately 0.5 mm, total length: 2.5 mm) was used to narrow both common carotid arteries and block most of the blood flow, thereby reducing the blood supply to the brain and putting the entire brain into a state of persistent chronic ischemia. The skin of the neck was sutured, and the mouse was returned to its cage and kept.

[0071] A total of three groups were set up, namely, a sham operation group (SHAM group), a model control group (BCAS group), and a drug candidate group (KLK1-PEG group).

[0072] Three days after BCAS surgery, mice were divided into groups and administered once a week for a total of seven times. The KLK1-PEG group was administered 80 μg / kg (KLK1 was hKLK1X1, and PEG was SPA10K), and the BCAS and SHAM groups were injected with the same volume of saline via the tail vein once a week.

[0073] On the 13th and 26th days after surgery, the Rotarod test was performed. The coordinated motor function of mice was evaluated by using a rotarod. A mouse was placed on a mouse rotarod (diameter 30 mm × length 60 mm) in the opposite direction to the direction of rotation. The rod was rotated and accelerated uniformly from 20 rpm / min to 40 rpm / min, and then the speed was maintained. The time that the mouse stayed on the rotarod (i.e., the time until the mouse fell off the rod) was recorded as an index. Each mouse was tested 3 to 5 times at intervals of 20 to 30 minutes. The average of the recorded values ​​was taken as the time until the mouse fell off, and the longer the time until the mouse fell off, the higher the coordinated motor function. On the 20th and 28th days after surgery, a wire hang test was performed to evaluate the damage to the forelimb muscle strength of the mice. The forelimbs of the mice were hung from a wire (1.5 mm in diameter x 60 cm in length), and the time the mouse was suspended from the wire (i.e., the time until the mouse fell from the wire) was recorded as an index. Each mouse underwent 3 to 5 tests at intervals of 20 to 30 minutes. The average of the recorded values ​​was taken as the time until the mouse fell, and the longer the time until the mouse fell, the less muscle damage was indicated.

[0074] On the 29th and 44th days after surgery, the mice were subjected to a Y-maze test to evaluate the damage to their working memory. The Y-maze is a Y-shaped test device with three identical arms connected at a 120° angle, each arm being 30 cm long, 8 cm wide, and 15 cm high. This is an experimental research device that evaluates learning and memory by utilizing the instinct of rodents to explore new environments. The mice were placed in the Y-maze and allowed to move freely. The order in which the mice entered each arm (all four paws entered the arms) was recorded for 7 minutes. The total number of times each arm was entered was defined as the number of arm entries, and the number of times each arm was entered consecutively was defined as the number of alternating behaviors. The alternating score was used as the evaluation index for the Y-maze test. Alternation score = [number of alternations / (number of arm entries-2)]*100 Animals that made fewer than eight arm entries during the experiment were excluded because their data did not accurately reflect changes. Higher alternation scores indicate less impairment of working memory.

[0075] On the 48th day after surgery, the Morris water maze test was performed to evaluate the damage to spatial learning and memory of the mice. The water temperature in the Morris water maze was maintained at 23±2°C, and the four positions for entering the water were indicated by writing "East," "South," "West," and "North" on the walls of the pool. The pool was divided into four equal parts, making four quadrants (E, S, W, and N). A video camera with a display system was installed above the water maze to synchronously record the mouse's behavioral trajectory. During the training period, the reference objects outside the water maze were not changed so that the mouse could identify the location of the platform. The latency to reach the platform (the time it takes for the mouse to reach the platform after entering the water) was used as a measurement index, and a shorter latency to reach the platform indicated less damage to spatial learning and memory.

[0076] 2. Results In comparison between the BCAS group and the SHAM group, statistically significant differences were observed in the results of the rotarod test, wire hang test, Y-maze test, and Morris water maze test between the two groups. This indicated that the creation of the BCAS model impaired the animals' motor coordination, forelimb muscle strength, working memory, and spatial learning and memory. Compared with the BCAS model group, the KLK1-PEG group showed improvement in each of the above experiments. Since the BCAS model is a well-accepted experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease, the results of the above experiments indicated that KLK1 or its derivatives can be used to treat cerebral small vessel disease and vascular cognitive impairment. Detailed results are summarized in Figures 3-9 and Tables 5-8.

[0077] [Table 5]

[0078] [Table 6]

[0079] [Table 7]

[0080] [Table 8]

[0081] Experiment 2 I. Group assignment and experimental design Bilateral common carotid artery spring constriction was performed to create a bilateral common carotid artery stenosis (BCAS) cerebral small vessel ischemic injury model in mice. The method for creating the BCAS cerebral small vessel injury model was the same as in Experiment 1.

[0082] A total of three groups were set up, namely, a model group (BCAS group), a KLK1 group, and a KLK1-PEG group, in which KLK1 is hKLK1X1 and PEG is SPA10K.

[0083] The subjects were divided into groups and administered the drugs 72 hours after BCAS. The KLK1 group received intravenous injections (0.5 mg / kg) five times a week, while the KLK1-PEG group received intramuscular injections (0.1 mg / kg) once a week for a total of 8 weeks.

[0084] After 8 weeks of administration, the mice were evaluated for their coordinated motor function using the Rotarod test. After 8 weeks of administration, the wire hang test was performed to measure the damage to the forelimb muscle strength of the mice. The evaluation method was the same as in Experiment 1. Eight weeks after administration, the Morris water maze test was performed to measure the extent of impairment of spatial learning and memory in the mice. The water surface of the Morris water maze was divided into four quadrants, and a platform was placed in the center of the third quadrant. In the experimental phase, the platform was removed from the water, and the time and distance that the animal spent in each quadrant were recorded. The proportion of the time and distance that the animal spent in the third quadrant to the total time and total distance indicates the animal's memory ability, and the higher the proportion, the better. This shows little damage to spatial learning and memory abilities.

[0085] 2. Results Statistically significant differences were observed in the results of the rotarod test, wire hang test, and Morris water maze test in the KLK1 group and KLK1-PEG group compared with the model group. KLK1 or KLK1-PEG can significantly improve the motor coordination, forelimb muscle strength, and spatial learning and memory disorders of animals created by the BCAS model, and can be used to treat cerebral small vessel disease and vascular cognitive impairment. Detailed results are summarized in Figures 10-13 and Tables 9-11.

[0086] [Table 9]

[0087] [Table 10]

[0088] [Table 11]

[0089] In the examples, an experimental animal model called the BCAS model, which is well accepted in the study of vascular cognitive dysfunction and cerebral small vessel disease, was used. By administering KLK1 or its polyethylene glycol modified form to BCAS model mice, the animals' impaired coordinated motor function, impaired muscle strength of the forelimbs, working memory, and spatial learning and memory functions were improved. This indicates that KLK1 or its polyethylene glycol modified form can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

[0090] In addition to the above test drugs, the hydrolysis of low molecular weight kininogen LMWK was catalyzed by the resilience of Other KLK1 derivatives that retain their biological function of releasing radikinin, such as other KLK1 mutants (hKLK1X2, hKLK1X3, hKLK1X4, etc.), partial proteins, fusion proteins, other PEG-modified forms, and other modified forms, all show similar effects when administered to BCAS model mice, namely, improving impaired coordination, impaired muscle strength in the forelimbs, working memory, and spatial learning and memory functions. These can also be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

Claims

1. Use of kallikrein 1 or a derivative thereof in the manufacture of a medicament for treating or ameliorating vascular cognitive dysfunction.

2. A pharmaceutical composition for treating or ameliorating vascular cognitive dysfunction, comprising kallikrein 1 or a derivative thereof.

3. A method for treating or ameliorating vascular cognitive impairment, comprising administering to a patient kallikrein 1 or a derivative thereof.

4. Use of kallikrein 1 or a derivative thereof in the manufacture of a medicament for treating or ameliorating post-stroke cognitive dysfunction.

5. A pharmaceutical composition for treating or ameliorating post-stroke cognitive dysfunction, comprising kallikrein 1 or a derivative thereof.

6. A method for treating or ameliorating post-stroke cognitive impairment, comprising administering to a patient kallikrein 1 or a derivative thereof.

7. Use of kallikrein 1 or a derivative thereof in the manufacture of a medicament for treating or ameliorating Alzheimer's disease accompanied by vascular lesions.

8. A pharmaceutical composition for treating or ameliorating Alzheimer's disease accompanied by vascular lesions, comprising kallikrein 1 or a derivative thereof.

9. A method for treating or ameliorating Alzheimer's disease accompanied by vascular lesions, comprising administering to a patient kallikrein 1 or a derivative thereof.

10. Use of kallikrein 1 or a derivative thereof in the manufacture of a medicament for treating or ameliorating cerebral small vessel disease or cognitive impairment due to cerebral small vessel disease.

11. A pharmaceutical composition for treating or ameliorating cerebral small vessel disease or cognitive impairment due to cerebral small vessel disease, comprising kallikrein 1 or a derivative thereof.

12. A method for treating or ameliorating cerebral small vessel disease or cognitive impairment due to cerebral small vessel disease, comprising administering kallikrein 1 or a derivative thereof to a patient.

13. The kallikrein 1 according to any one of claims 1 to 12 is characterized in that it is native human tissue kallikrein 1 or is a mutated recombinant human tissue kallikrein 1 or a non-mutated recombinant human tissue kallikrein 1.

14. The kallikrein 1 derivative according to any one of claims 1 to 12 is characterized in that it is a full-length protein of kallikrein 1, a partial protein thereof, or a mutant, fusion protein, or various forms of modified form thereof.

15. The kallikrein 1 derivative according to any one of claims 1 to 12, characterized in that it is a polyethylene glycol modified form of kallikrein 1.

16. The polyethylene glycol modified form of kallikrein 1 according to claim 15 is M-SPA- A polyethylene glycol-modified kallikrein 1, characterized in that it is modified with M-SPA-5K, M-SPA-10K, Y-PALD-30K, or Y-PALD-40K.

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