WO2023142889A1 - 激肽释放酶i或其衍生物在治疗vci、psci或csvd中的应用 - Google Patents

激肽释放酶i或其衍生物在治疗vci、psci或csvd中的应用 Download PDF

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WO2023142889A1
WO2023142889A1 PCT/CN2022/144054 CN2022144054W WO2023142889A1 WO 2023142889 A1 WO2023142889 A1 WO 2023142889A1 CN 2022144054 W CN2022144054 W CN 2022144054W WO 2023142889 A1 WO2023142889 A1 WO 2023142889A1
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kallikrein
derivatives
treating
improving
cognitive dysfunction
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马永
江辰阳
王俊
王和
许振�
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江苏众红生物工程创药研究院有限公司
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Publication of WO2023142889A1 publication Critical patent/WO2023142889A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21106—Hepsin (3.4.21.106)

Definitions

  • the present invention relates to the application of kallikrein I or its derivatives in the treatment of vascular cognitive impairment (VCI), post-stroke cognitive impairment (PSCI) or cerebral small vessel disease (CSVD).
  • VCI vascular cognitive impairment
  • PSCI post-stroke cognitive impairment
  • CSVD cerebral small vessel disease
  • VCI vascular cognitive impairment
  • obvious cerebrovascular disease such as cerebral infarction and cerebral hemorrhage, etc.
  • non-obvious cerebrovascular diseases such as leukoaraiosis, chronic cerebral ischemia
  • VCI cognitive impairment Patients with vascular cognitive impairment experience cognitive impairment and psychobehavioral symptoms. In some patients, the impairment of cognitive function is mainly in abstract thinking, concept formation and conversion, thinking flexibility, information processing speed and other functional impairments, while memory is relatively preserved; in some patients, multi-domain impairments may also be evident in memory impairment.
  • Drugs for VCI cognitive impairment include cholinesterase inhibitors and non-competitive N-methyl-D-asparagine receptor antagonists.
  • Psychological and behavioral symptoms include abnormal perception, thinking, mood, and behavior, etc. Common apathy, depression, irritability, agitation/aggression, targeted treatment drugs, such as antidepressant drugs 5-HT reuptake inhibitors, etc. (“Chinese Vascular Cognitive Impairment Guidelines for diagnosis and treatment").
  • Post-stroke cognitive impairment (PSCI) cognitive impairment caused by cerebral small vessel disease (CSVD), Alzheimer's disease (AD) with vascular disease ) etc. are important subtypes of VCI.
  • Cognitive impairment after stroke emphasizes that stroke triggers cognitive impairment, which also involves cognitive impairment and psychobehavioral symptoms.
  • Drug treatment generally uses acetylcholinesterase inhibitors and non-competitive N-methyl-D-asparagine receptor antagonists (memantine); drug treatment for post-stroke depression generally uses antidepressants.
  • the latest "2021 ESO/EAN Joint Guidelines: Post-Stroke Cognitive Impairment” issued by the European Stroke Organization states that there is no drug treatment for post-stroke cognitive impairment. Only one trial focused specifically on post-stroke cognitive impairment, but this was not positive. The guideline also points out that high-quality data from randomized controlled trials for PSCI are clearly insufficient.
  • Cerebral small vessel disease is a series of clinical, imaging, and pathological syndromes caused by various risk factors affecting cerebral arterioles, arterioles, capillaries, venules, and venules. It is divided into acute CSVD and chronic CSVD. Acute CSVD can lead to ischemic stroke. At present, it is recommended to refer to the prevention and treatment of acute ischemic stroke, such as antihypertensive, thrombolytic, antiplatelet, and lipid-lowering treatments. Patients with chronic CSVD may experience symptoms such as cognitive impairment, motor impairment, emotional impairment, and defecation disorders. Symptomatic treatment is generally recommended after a definite diagnosis.
  • cholinesterase inhibitors and non-competitive N-methyl-D-asparagine receptor antagonists can be selected for cognitive impairment caused by CSVD, and antidepressants such as serotonin reuptake inhibitors are generally used for depression ( "Chinese Expert Consensus on Diagnosis and Treatment of Cerebrovascular Disease").
  • the first technical problem solved by this application is to provide a new drug for treating vascular cognitive dysfunction. Specifically, it provides the application of kallikrein I or its derivatives in the preparation of medicines for treating or improving vascular cognitive dysfunction.
  • the present invention also provides a pharmaceutical composition for treating or improving vascular cognitive dysfunction, comprising kallikrein I or its derivatives.
  • the present invention also provides a method for treating or improving vascular cognitive dysfunction, that is, administering kallikrein I or its derivatives to patients.
  • the vascular cognitive dysfunction includes, but is not limited to, post-stroke cognitive dysfunction, cognitive dysfunction caused by cerebral small vessel disease, Alzheimer's disease with vascular lesions, and the like.
  • said vascular cognitive impairment is post-stroke cognitive impairment.
  • the vascular cognitive dysfunction is cognitive dysfunction caused by cerebral small vessel disease.
  • the vascular cognitive dysfunction is Alzheimer's disease with vascular lesions.
  • the second technical problem solved by this application is to provide a new drug for treating cerebral small vessel disease. Specifically, it provides the application of kallikrein I or its derivatives in the preparation of medicines for treating or improving cerebral small vessel diseases.
  • the present application also provides a pharmaceutical composition for treating or improving cerebral small vessel disease, comprising kallikrein I or its derivatives.
  • the present application also provides a method for treating or improving cerebral small vessel disease, that is, administering kallikrein I or its derivatives to patients.
  • the kallikrein I may be a naturally extracted kallikrein I or a recombinant kallikrein I.
  • the kallikrein I derivatives may be full-length proteins, partial proteins, mutants, fusion proteins, and modifications of various forms of kallikrein I.
  • the kallikrein I derivative is polyethylene glycol-modified kallikrein I.
  • the kallikrein I or its derivatives can be administered alone, or in combination with other drugs for the treatment or improvement of cerebral small vessel disease and vascular cognitive dysfunction, such as cholinesterase inhibitors, non-competitive N-methyl-D-asparagine receptor antagonists, etc.
  • Administration methods include but are not limited to injection administration, oral administration and the like.
  • the injection administration methods include but not limited to intravenous injection, subcutaneous injection, intramuscular injection and the like.
  • the more recognized animal models of vascular cognitive impairment include: four-vessel occlusion (4-VO) method for rats, improved 4-VO method, three-stage 4-VO method, and two-vessel occlusion method (2-vessel occlusion, 2-VO) method, modified 2-VO method, and modified common carotid artery occlusion (modified common carotid artery occlusion, mCCAO) on one side of common carotid artery occlusion, etc.; Arterial stenosis (bilateral CCA stenosis, BCAS) and asymmetric carotid artery surgery (asymmetric CCA surgery, ACAS), etc. (“Research progress in animal models of vascular cognitive impairment").
  • Recognized animal models of cerebral small vessel disease mainly include bilateral common carotid artery ligation model, bilateral common carotid artery stenosis model (BCAS), stroke susceptibility spontaneously hypertensive rat model, etc. consensus").
  • BCAS bilateral common carotid artery stenosis model
  • stroke susceptibility spontaneously hypertensive rat model etc. consensus
  • kallikrein I or its derivatives can significantly improve impaired motor coordination, forelimb damaged muscle strength, working memory, and spatial learning and memory functions. Therefore kallikrein I or its derivatives can be used for the treatment of vascular cognitive dysfunction and cerebral small vessel disease.
  • post-stroke cognitive impairment cognitive impairment caused by cerebral small vessel disease
  • Alzheimer's disease with vascular lesions are all important subtypes of vascular cognitive impairment.
  • treatment guidelines or expert consensus for cerebral small vessel disease generally recommend the use of acetylcholinesterase inhibitors, Noncompetitive N-methyl-D-asparagine receptor antagonist (memantine); antidepressants are generally recommended for pharmacological treatment of psychobehavioral symptoms such as depression. Therefore, kallikrein I or its derivatives can be used to treat various subtypes of vascular cognitive dysfunction, such as post-stroke cognitive dysfunction, cognitive dysfunction caused by cerebral small vessel disease, and Alzheimer's disease with vascular disease. Alzheimer's disease, etc.
  • the kallikrein-kinin system (KKS, Kinin-kallikrein system) is involved in a variety of physiological and pathological processes, such as the regulation of cardiovascular, renal and nervous system functions.
  • the KKS system includes kallikrein, kininogen, kinin, kinin receptors (B1, B2 receptors) and kininase.
  • Kallikrein also known as kininogenase or kallikrein, is a serine protease that is divided into two major classes: plasma kallikrein I (PK) and tissue kallikrein I (TK), both of which exert very important physiological function.
  • human tissue kallikrein is composed of at least 15 members (KLK1-KLK15), among which there are more studies on tissue kallikrein I (KLK1), and KLK1 acts on the corresponding Receptors play a series of biological roles.
  • KLK1-KLK15 tissue kallikrein I
  • KLK1 acts on the corresponding Receptors play a series of biological roles.
  • Two types of kallikrein I for injection have been launched in China, and the indications include microcirculatory disorders and mild to moderate acute ischemic stroke.
  • vascular cognitive impairment, cerebral small vessel disease, etc. are quite different from the approved indications of Kallikrein I.
  • acute ischemic stroke is based on vascular wall lesions caused by various reasons.
  • the main or branch arterial lumen of the cerebral artery is narrowed, occluded or thrombosed, resulting in a decrease in blood flow or interruption of blood supply in the brain, causing Cerebral tissue ischemia, hypoxic necrosis, and focal neurological symptoms and signs.
  • VCI refers to the risk factors of cerebrovascular disease (such as hypertension, diabetes, hyperlipidemia, etc.), obvious (such as cerebral infarction and cerebral hemorrhage, etc.) or insignificant cerebrovascular disease (such as leukoaraiosis, chronic cerebral ischemia) caused A broad class of syndromes ranging from mild cognitive impairment to dementia.
  • Neurovascular unit (NVU) dysfunction plays an important role in the pathogenesis of cerebral small vessel disease. Any structural or functional change of NVU can lead to CSVD.
  • Common mechanisms include chronic cerebral ischemia and hypoperfusion, endothelial function Different mechanisms interact with each other, such as blood-brain barrier damage, interstitial fluid reflux disorder, inflammatory response, and genetic factors. (the neurovaseular unit coming of age: a journey through neurovaseular coupling in health and disease)
  • vascular cognitive impairment and cerebral small vessel disease are quite different from those of vascular cognitive impairment and cerebral small vessel disease.
  • the most effective treatment for acute ischemic stroke is vascular recanalization within the time window, including intravenous thrombolysis, mechanical thrombectomy, angioplasty, etc.
  • the success rate of treatment is closely related to the time of onset.
  • Drug therapy includes antiplatelet, anticoagulation, fiber reduction, volume expansion, vasodilation, statins, neuroprotective drugs, etc.
  • Cognitive impairment and psychobehavioral symptoms usually occur in VCI and PSCI.
  • Drugs for cognitive impairment in VCI and PSCI include cholinesterase inhibitors and non-competitive N-methyl-D-asparagine receptor antagonists , Drugs for the targeted treatment of mental and behavioral symptoms, such as antidepressants such as serotonin reuptake inhibitors.
  • Patients with chronic cerebrovascular disease may experience symptoms such as cognitive impairment, motor impairment, emotional impairment, and defecation disorders. It is generally recommended to carry out symptomatic treatment after a clear diagnosis.
  • cognitive impairment caused by CSVD can be treated with cholinesterase inhibitors and non-competitive N-methyl-D-asparagine receptor antagonists, and antidepressants such as serotonin reuptake inhibitors are usually used for depression.
  • kallikrein I or its derivatives can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, cerebral small vessel disease and the like. It is quite different from the approved indications of kallikrein I, and provides a new method for the treatment of vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.
  • FIG. 1 KLK1 and its mutants activate phosphorylation of downstream CREB proteins.
  • Figure 5 Effect of drugs on the wire hang time on the 20th day of Experiment 1.
  • Figure 7 The effect of drugs on the Y Maze rotation score on the 29th day of Experiment 1.
  • Figure 8 The effect of drugs on the Y Maze rotation score on the 44th day of Experiment 1.
  • Figure 9 Effect of drugs on the latency period of Morris maze on the 48th day of Experiment 1.
  • Figure 10 The effect of the drugs in Experiment 2 on the Rotarod test.
  • Figure 12 Effects of Experiment 2 drugs on Morris maze stage latency (movement distance in quadrant III).
  • Figure 13 Effect of Experiment 2 drugs on Morris maze stage latency (time to be active in quadrant III).
  • hKLK1 Unmutated human tissue kallikrein I sequence identical to natural human tissue kallikrein I; covers various homologues of human KLK1, including but not limited to Genbank accession numbers AAA59455.1, NP002248.1 , AAA36136.1, AAP35917, AAU12569, etc. as shown in KLK1. In a specific embodiment, the sequence is shown in SEQ ID NO: 5.
  • the three glycosylation modification sites N78, N84 and N141 of KLK1 refer to asparagine at positions 78, 84 and 141 of the amino acid sequence of KLK1, respectively.
  • the corresponding N-glycosylation triad subsequence motifs are NMS, NHT, NFS respectively, N stands for asparagine, M stands for methionine, S stands for serine, H stands for histidine, T stands for threonine, F stands for Phenylalanine.
  • Unmutated hKLK1 has high glycosylation modification, that is, there are more glycosylation modifications at the glycosylation modification sites at N78, N84, and N141.
  • Unmutated hKLK1 has low glycosylation modification, that is, there are more sugar modifications at the glycosylation modification sites at N78 and N84, and there is no glycosylation modification at the glycosylation modification site at N141 , or only a small amount of glycosylation modification occurs.
  • Recombinant highly glycosylated hKLK1 and low glycosylated hKLK1 can be separated by conventional purification methods, such as hydrophobic chromatography, anion chromatography, cation chromatography or a combination thereof. Applicants have unexpectedly discovered that the activity of hypoglycosylated KLK1 is much higher than that of hyperglycosylated KLK1.
  • PEG-hKLK1 (highly glycosylated): PEG-modified hyperglycosylated hKLK1, the hyperglycosylated hKLK1 refers to the above-mentioned unmutated hKLK1 with hyperglycosylation modification.
  • PEG-hKLK1 (low glycosylation): PEG-modified low glycosylated hKLK1, the low glycosylated hKLK1 refers to the above-mentioned unmutated hKLK1 with low glycosylation modification.
  • KLK1 is an enzyme with abundant glycosylation modification
  • N-glycosylation modification is the most concerned glycosylation modification in glycoprotein drugs
  • the sugar chain is connected with the free -NH2 group of specific asparagine in the nascent peptide chain .
  • the triple subsequence motif of the N-glycosylation modification site must be NXS or NXT, where N represents asparagine, S represents serine, T represents threonine, X represents other amino acids except proline, and does not satisfy Asparagine under the above conditions does not undergo N-glycosylation modification.
  • hKLK1X1, hKLK1X2, hKLK1X3, and hKLK1X4 represent different mutants, and their sequences are shown in SEQ ID NO: 1, 2, 3, and 4, respectively. They carried out point mutations on the glycosylation modification site N141, and mutated asparagine into neutral polar amino acid glutamine (Gln), acidic amino acid aspartic acid (Asp), and basic amino acid arginine ( Arg), aliphatic amino acid alanine (Ala) four different types of amino acids, the mutant has no NFS N-glycosylation motif, and only two glycosylation modification sites, so as to achieve more uniform products and low sugar groups.
  • the results showed that hKLK1 had a higher yield, and it was surprisingly found that the activity of mutated low-glycosylated KLK1 was much higher than that of unmutated low-glycosylated KLK1.
  • N at NFS i.e. N141
  • F at NFS i.e. F142
  • proline 0 in N and S 1 or 2 amino acids are mutated to any other amino acid
  • S i.e.
  • PEG-hKLK1X PEG-modified hKLK1 mutant.
  • KLK1 derivatives including both KLK1 full-length protein and partial KLK1 protein; including KLK1 mutants of this application, as well as proteins and fusion proteins obtained by further mutations on the basis of KLK1 mutants of this application (including but not limited to albumin fusion, Fc fusion, etc.), various forms of modifications (including but not limited to PEGylation modifications).
  • Polyethylene glycol usually polymerized by ethylene oxide, has branched, linear and multi-armed types. Generally, those with a molecular weight below 20,000 are called PEG, and those with a higher molecular weight are called PEO. Ordinary polyethylene glycol has a hydroxyl group at both ends, and if one end is blocked with a methyl group, methoxy polyethylene glycol (mPEG) can be obtained.
  • mPEG methoxy polyethylene glycol
  • PEG modification agent refers to polyethylene glycol derivatives with functional groups, which are activated polyethylene glycol and can be used for protein and polypeptide drug modification.
  • the polyethylene glycol modifier used in this application was purchased from Jiangsu Zhonghong Bioengineering Pharmaceutical Research Institute Co., Ltd. or Beijing Jiankai Technology Co., Ltd.
  • the actual molecular weight of the PEG modifier with a specific molecular weight can be 90%-110% of the marked value, for example, the molecular weight of PEG5K can be 4.5kDa-5.5kDa.
  • the PEG5K used in the examples specifically refers to M-SPA-5K, which is a linear monomethoxypolyethylene glycol succinimide propionate with a molecular weight of about 5 kDa.
  • the structure is as shown in formula (1), and n is from 105 to an integer of 110,
  • the PEG10K used in the examples specifically refers to M-SPA-10K, which is a linear monomethoxypolyethylene glycol succinimide propionate with a molecular weight of about 10 kDa.
  • the structure is as shown in formula (1), and n is from 220 to An integer of 225.
  • the PEG30K used in the examples specifically refers to Y-PALD-30K, which is a branched polyethylene glycol propionaldehyde with a molecular weight of about 30kD.
  • the structure is shown in formula (2), and m is an integer from 335 to 340.
  • the PEG40K used in the examples specifically refers to Y-PALD-40K, which is a branched polyethylene glycol propionaldehyde with a molecular weight of about 40 kD.
  • the structure is shown in formula (2), and m is an integer ranging from 450 to 455.
  • Suitable polyethylene glycol modifiers for KLK1 are not limited to the above-mentioned polyethylene glycol modifiers used in specific examples, and other known polyethylene glycol modifiers can also be tried.
  • the PEGylated modification of KLK1 used in the following examples is a PEGylated protein obtained by performing PEGylation and purification using methods well known in the art. For example: it can be prepared by referring to Chinese patent CN109498815A, Chinese patent CN107760661A or the prior application CN202111353294.2.
  • Kallikrein I catalyzes the hydrolysis of low-molecular-weight kininogen LMWK in vivo to release lysyl bradykinin to perform biological functions.
  • the hydrolysis reaction involves the cleavage of the peptide bond at the carboxyl terminal of arginine (Arg). Therefore, p-nitroaniline (PNA) can be generated based on the cleavage of the amide bond between Arg and p-nitroaniline in the hydrolyzed synthetic chromogenic substrate S-2266 (H-D-Val-Leu-Arg-PNA), and the detection of PNA at 405nm to evaluate the in vitro biological activity of recombinant hKLK1 and its mutants.
  • PNA p-nitroaniline
  • the activity unit IU is defined as the amount of enzyme that hydrolyzes 1 ⁇ mol S-2266 into PNA per minute at 37°C and pH 8.0, which is 1 IU.
  • the reaction system is 200 ⁇ l 20mM Tris buffer solution, 10 ⁇ l test sample, 20 ⁇ l 20mM S-2266 substrate solution, placed in a 37°C water bath for accurate reaction for 10min, and 20 ⁇ l 50% acetic acid solution was added to terminate the reaction, based on different concentrations
  • the standard curve fitted by the PNA standard was used to quantify the amount of PNA produced in the reaction system.
  • the in vitro biological activity detection of hKLK1 and hKLK1 mutants with high and low glycosylation was completed by using the above method.
  • KLK1 exerts biological functions in vivo by catalyzing the hydrolysis of low molecular weight kininogen LMWK to release lysyl bradykinin.
  • This example compares the situation that the effector molecule bradykinin is generated by the enzymatic reaction under different ratios of the substrate (low molecular kininogen LMWK) and the enzyme (KLK1 or its PEG modification). The effector molecules were separated, the peak area of the product was calculated, the generation curve of bradykinin was drawn under different ratios of substrate and enzyme, and the amount of effector molecules generated by different test samples under the same reaction conditions were compared. Through this in vitro simulation in vivo Mode of action means to indirectly compare the in vivo effects of different test samples.
  • Kb is KLK1 extracted from porcine pancreas
  • PEG-Kb is Kb modified with PEG10K
  • PEG10K-hKLK1X1 and PEG-Kb can be prepared according to the conventional methods of modifying drugs with polyethylene glycol.
  • the chromatographic peaks whose retention time is 13 ⁇ 0.5min (bradykinin peak position) in the reaction mixture chromatogram are integrated and summed, and compared with the unit mass (1mg/ml, 10 ⁇ l loading amount) bradykinin peak area comparison, calculate the concentration of the bradykinin generated by the enzymatic reaction, and calculate the amount of bradykinin generation according to the total reaction system in Table 2, finally converted into the condition of each molar ratio
  • the amount of bradykinin produced per milligram of enzyme ( ⁇ g/mg) are shown in Table 4.
  • the detection results based on natural substrates show that in the overall trend, the bradykinin produced by the enzymatic reaction of the modified protein is lower than that of the sample before modification, which is consistent with the consistent characteristics of PEG-modified proteins.
  • the PEG-modified protein can Maintain the enzymatic process more gently to achieve the sustained release of effector molecules; at the same time, the onset of KLK1 drugs is based on the regulation of the KKS system in the body. This regulation includes the release and clearance of effector molecules. Obviously, a mild and sustained enzyme The promotion process can effectively reduce the clearance mechanism of the KKS system, so that PEG-modified KLK1 can play a more stable and effective biological role.
  • CHO-K1 cells (containing B2 receptors) in the logarithmic growth phase were collected, and live cell counts were performed with a hemocytometer.
  • the live cell suspension was adjusted to 3 ⁇ 10 6 cells/mL with the medium and inoculated on a 6-well cell culture plate, with a final cell concentration of 3 ⁇ 10 5 cells/well.
  • the cells were cultured in a CO 2 incubator with 5.0% CO 2 at 37.0°C for 24-48 hours. When the cell density in each well reaches more than 90%, wash 2 times with PBS. Prepare samples with F12 medium in advance (KLK1 and LMWK are mixed at a mass ratio of 1:10, the reaction system is F12 medium, and react for 15 minutes).
  • Cyclic adenosine monophosphate response element binding protein (cAMP-response element binding protein, CREB) is a signaling molecule directly acting downstream after B2R activation.
  • the level of ⁇ can reflect the ability of the test substance to activate B2R to a certain extent, and at the same time reflect the significance of the neuroprotective effect from the side.
  • KLK1 can catalyze the production of low-molecular-weight kininogen LMWK.
  • the active molecule binds to the B2 receptor, it activates the activation of the downstream signal CREB protein.
  • it can activate the downstream CREB protein. Phosphorylation is consistent with the mechanism of action of the drug, and the in vitro biological activity of the sample is qualitatively evaluated by judging the phosphorylation level of the downstream CREB protein.
  • the CHO-K1 (containing B2 receptor) cells in the logarithmic growth phase were collected, and the living cells were counted with a hemocytometer.
  • the live cell suspension was adjusted to 3 ⁇ 10 6 cells/mL with medium and inoculated on a 6-well cell culture plate, with a final cell concentration of 3 ⁇ 10 5 cells/well.
  • the cells were cultured in a CO 2 incubator with 5.0% CO 2 at 37.0°C for 24-48 hours. When the cell density in each well reaches more than 90%, wash 2 times with PBS.
  • the sample (Lys-BK) was prepared in advance with F12 medium, 1ml of sample was added to each well, the final concentration of Lys-BK was 1 ⁇ M, and the control group was F12 medium.
  • the cell culture plate was placed in a 37.0°C, 5.0% CO 2 incubator and incubated for 0.5h. After the incubation, the 6-well plate was taken out of the incubator, washed twice with PBS, and the PBS was aspirated for the last time.
  • Extracellular regulated protein kinases 1/2 (ERK1/2) and cyclic adenosine monophosphate response element binding protein (cAMP-response element binding protein, CREB) are signaling molecules directly acting downstream after B2R activation, CREB
  • ERK1/2 Extracellular regulated protein kinases 1/2
  • cAMP-response element binding protein CREB
  • the activation of ERK1/2 can prevent the occurrence of inflammatory response and neuron damage after ischemia, so the level of its expression can reflect the ability of the test substance to activate B2R to a certain extent, and at the same time reflect the significance of the neuroprotective effect from the side.
  • Lys-BK Based on the B2-NFAT-CHO-K1 cell line, the combination of lysyl bradykinin Lys-BK and B2 receptor activates the activation of downstream signal CREB and p-ERK1/2 protein, as shown in Figure 2, Lys-BK It can activate the phosphorylation of downstream CREB and p-ERK1/2 proteins.
  • Bilateral common carotid artery stenosis (BCAS) cerebral small vessel ischemic injury model in mice was established by bilateral common carotid artery spring contraction method.
  • the BCAS cerebral small vessel ischemic injury model was used to achieve persistent chronic ischemia of the forebrain in mice.
  • the mice were anesthetized with isoflurane. First, the mice were placed in the induction box of the anesthesia machine to induce anesthesia, then the mice were placed in a supine position and connected to a breathing mask, the skin was prepared and disinfected, the neck was cut midline, and the bilateral necks were separated.
  • Common artery use custom-made spring (spring material: imported piano wire, size: wire diameter: 0.08mm, inner diameter: 0.18mm, pitch: about 0.5mm, total length: 2.5mm) to shrink the bilateral common carotid arteries and block the large Part of the blood flow, resulting in decreased blood supply to the brain, persistent chronic ischemia of the whole brain.
  • the neck skin was sutured, disinfected, and put back into the cage for rearing.
  • the administration began in groups on the third day after BCAS, once a week, for a total of 7 times.
  • the KLK1-PEG group 80 ⁇ g/kg, the KLK1 is hKLK1X1, and the PEG is SPA10K
  • the BCAS group and the SHAM group were injected with an equal volume of normal saline once a week through the tail vein.
  • Rotarod test was used to detect the motor coordination function of mice on the 13th and 26th day after operation. Place the mouse on the mouse rotarod (30mm diameter x 60mm length) in the opposite direction of rotation, start the rotarod, accelerate uniformly at 20rpm/min to 40rpm/min and then maintain it. Record the mouse's time on the bar (ie, the mouse's fall time) as an indicator. Each mouse needs to be tested 3 to 5 times with an interval of 20 to 30 minutes. The average value was taken as the mouse rotarod drop time, and the longer the rotarod drop time, the better the motor coordination function.
  • the wire hang test was used to detect the impairment of the forelimb muscle strength of the mice on the 20th day and 28th day after operation.
  • the mouse's forelimbs were hung on a steel wire (1.5 mm diameter x 60 cm long), and the time the mouse hung on the steel wire (ie, the time the mouse fell) was recorded as an index. It needs to be detected 3 to 5 times, each interval is 20 to 30 minutes, and the average value is taken as the wire drop time of the mouse. The longer the wire falls, the less muscle damage.
  • the impairment of the working memory of the mice was detected on the 29th and 44th day after operation using the Y Maze test.
  • the Y-maze is a "Y-shaped" experimental device composed of three identical arms at 120° to each other. Each arm is 30cm long, 8cm wide, and 15cm high. It uses the nature of rodents to explore new environments and is used for learning and memory.
  • the water maze test (Morris maze) was used to detect the impairment of spatial learning and memory in mice on the 48th day after operation.
  • the water temperature in the water maze is maintained at (23 ⁇ 2)°C, four water entry points are marked on the wall of the pool, the pool is divided into four quadrants (E, S, W, N), and a camera with a display system is placed above the maze , for synchronous recording of mouse movement trajectories.
  • the external reference of the maze was kept constant during training to allow the rats to locate the platform. Taking the stage latency (the time from entering the water to finding the platform) as the detection index, the shorter the stage latency, the less the impairment of spatial learning and memory.
  • the BCAS model is a relatively recognized experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease. The above experimental results indicate that KLK1 or its derivatives can be used to treat cerebral small vessel disease and vascular cognitive impairment.
  • Bilateral common carotid artery stenosis (BCAS) cerebral small vessel ischemic injury model in mice was established by bilateral common carotid artery spring contraction method.
  • the model group (BCAS group), the KLK1 group, and the KLK1-PEG group (the KLK1 is hKLK1X1, and the PEG is SPA10K).
  • KLK1 group 0.5 mg/kg was administered intravenously five times a week
  • KLK1-PEG group 0.1 mg/kg was administered intramuscularly once a week for a total of 8 weeks.
  • Rotarod test was used to detect the motor coordination function of mice after 8 weeks of administration, and the evaluation method was the same as that in Experiment 1.
  • the wire hang test (Wire hang) was used to detect the impairment of the forelimb muscle strength of the mice after 8 weeks of administration, and the evaluation method was the same as that of Experiment 1.
  • Water maze test (Morris maze) was used to detect the impairment of spatial learning and memory in mice after 8 weeks of administration, the water surface was divided into 4 quadrants, and the platform was placed in the center of the III quadrant; the platform in the water was removed during the test phase, Record the time and distance of animals in different quadrants; the ratio of the time and distance of animals in quadrant III to the total time and total distance reflects the animal's memory ability, and the higher the ratio, the milder the impairment of spatial learning and memory.
  • KLK1 group, KLK1-PEG group had significant statistical differences in the results of rotarod test, hanging rope test, and water maze test.
  • KLK1 or KLK1-PEG can significantly improve the movement coordination of animals induced by BCAS modeling , forelimb muscle strength, spatial learning and memory impairment, can be used to treat cerebral small vessel disease, vascular cognitive impairment.
  • the results are shown in Figures 10-13 and Tables 9-11.
  • * indicates P ⁇ 0.05 compared with the model group.
  • * indicates P ⁇ 0.05 compared with the model group.
  • the embodiment adopts a relatively recognized experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease, that is, the BCAS model.
  • the BCAS model mice are given KLK1 or its polyethylene glycol modification, the impaired movement can be improved.
  • Coordination function, forelimb damaged muscle strength, working memory, and spatial learning and memory functions can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.
  • BCAS model mice were given other KLK1 derivatives that better retained KLK1 to catalyze the hydrolysis of low-molecular-weight kininogen LMWK to release lysyl bradykinin to exert biological functions, such as other mutants of KLK1 (hKLK1X2, hKLK1X3, hKLK1X4, etc.), partial proteins, fusion proteins, other PEG modifiers, and other forms of modifiers, all can observe similar effects, that is, to improve the impaired motor coordination function of BCAS model mice, the strength of forelimb damaged muscles, work Memory and spatial learning and memory functions can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

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Abstract

本发明涉及激肽释放酶Ⅰ或其衍生物在治疗血管性认知功能障碍(VCI)、卒中后认知功能障碍(PSCI)或脑小血管病(CSVD)中的应用。

Description

激肽释放酶I或其衍生物在治疗VCI、PSCI或CSVD中的应用 技术领域
本发明涉及激肽释放酶I或其衍生物在治疗血管性认知功能障碍(VCI)、卒中后认知功能障碍(PSCI)或脑小血管病(CSVD)中的应用。
背景技术
随着社会发展和人口老龄化，脑血管病的发病率逐年升高，严重影响了患者和家属的生活质量。1993年Hachinski和Bowler首次提出了血管性认知功能障碍(vascular cognitive impairment，VCI)的概念，它是指由脑血管病危险因素(如高血压、糖尿病、高血脂等)、明显的脑血管病(如脑梗死和脑出血等)或不明显的脑血管病(如白质疏松、慢性脑缺血)引起的从轻度认知障碍到痴呆的一大类综合征。(《2019年中国血管性认知障碍诊治指南》、《中国脑卒中防治指导规范(2021版)》之《中国血管性认知损害诊疗指导规范》)
血管性认知功能障碍患者会出现认知功能损害和精神行为症状。部分患者的认知功能损害以抽象思维、概念的形成和转换、思维灵活性、信息处理速度等功能损害为主，而记忆力相对保留；部分患者表现为多领域障碍，记忆力亦可明显受损。针对VCI认知功能损害的药物有胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂。精神行为症状包括知觉、思维、心境、行为异常等，常见淡漠、抑郁、烦躁、激越/攻击，针对性治疗药物，如抗抑郁药物5-羟色胺再摄取抑制剂等(《中国血管性认知损害诊疗指导规范》)。卒中后认知功能障碍(post-stroke cognitive impairment，PSCI)、脑小血管病(cerebral small vessel disease，CSVD)导致的认知功能障碍、伴有血管病变的阿尔兹海默病(Alzheimer’s disease，AD)等均是VCI的重要亚型。
卒中后认知功能障碍强调卒中触发认知功能障碍，同样涉及认知功能损害和精神行为症状。考虑到PSCI、VCI和AD在神经病理和神经生化机制方面存在一定重叠性，中国《卒中后认知障碍管理专家共识2021》建议参考VCI、AD等相关的研究和证据，即针对认知障碍的药物治疗一般采用乙酰胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂(美金刚)；针对卒中后抑郁的药物治疗 一般采用抗抑郁药。欧洲卒中组织最新发布的“2021ESO/EAN联合指南:卒中后认知障碍”指出：尚无药物治疗卒中后认知障碍。只有一项试验特别关注卒中后认知障碍，但并未有阳性结果。该指南同时指出针对PSCI的随机对照试验的高质量数据明显不足。
脑小血管病是由多种危险因素影响脑内小动脉、微动脉、毛细血管、微静脉和小静脉所导致的一系列临床、影像学和病理综合征，分为急性CSVD、慢性CSVD。急性CSVD可导致缺血性卒中，目前建议参考急性缺血性卒中的防治方案，如采取降压、溶栓、抗血小板、降脂治疗。慢性CSVD患者可出现认知障碍、运动障碍、情感障碍和二便障碍等症状，一般建议在明确诊断后进行对症处理。如CSVD导致的认知障碍可选择胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂，抑郁一般选用抗抑郁药物如5-羟色胺再摄取抑制剂等(《中国脑小血管病诊治专家共识》)。
发明内容
本申请解决的第一个技术问题是提供一种新的治疗血管性认知功能障碍的药物。具体而言提供了激肽释放酶I或其衍生物在制备治疗或改善血管性认知功能障碍的药物中的应用。
本发明还提供一种用于治疗或改善血管性认知功能障碍的药物组合物，包含激肽释放酶I或其衍生物。
本发明还提供一种治疗或改善血管性认知功能障碍的方法，即给予患者激肽释放酶I或其衍生物。
所述的血管性认知功能障碍包括但不限于卒中后认知功能障碍、脑小血管病导致的认知功能障碍、伴有血管病变的阿尔兹海默病等。
优选地，所述血管性认知功能障碍是卒中后认知功能障碍。
优选地，所述血管性认知功能障碍是脑小血管病导致的认知功能障碍。
优选地，所述血管性认知功能障碍是伴有血管病变的阿尔兹海默病。
本申请解决的第二个技术问题是提供一种新的治疗脑小血管病的药物。具体而言提供了激肽释放酶I或其衍生物在制备治疗或改善脑小血管病的药物中的应用。
本申请还提供一种用于治疗或改善脑小血管病的药物组合物，包含激肽释放酶I或其衍生物。
本申请还提供一种治疗或改善脑小血管病的方法，即给予患者激肽释放酶I或其衍生物。
所述激肽释放酶I可以是天然提取的激肽释放酶I或重组激肽释放酶I。所述激肽释放酶I衍生物可以是激肽释放酶I的全长蛋白、部分蛋白、突变体、融合蛋白、各种形式修饰物。
优选地，所述激肽释放酶I衍生物是聚乙二醇修饰的激肽释放酶I。
所述激肽释放酶I或其衍生物可以单独给药，也可以联合其他治疗或改善脑小血管病、血管性认知功能障碍的药物给药，如胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂等。
给药方式包括但不限于注射给药、口服给药等。所述注射给药方式包括但不限于静脉注射、皮下注射、肌肉注射等。
血管性认知功能障碍比较公认的动物模型有：针对大鼠的四血管闭塞法(4-vessel occlusion，4-VO)法、改良4-VO法、3期4-VO法、两血管闭塞法(2-vessel occlusion，2-VO)法、改良2-VO法、以及一侧颈总动脉闭塞一侧颈总动脉狭窄改良法(modified common carotid artery occlusion，mCCAO)等；针对小鼠的颈总动脉狭窄(bilateral CCA stenosis，BCAS)和不对称颈动脉手术(asymmetric CCA surgery，ACAS)等(《血管性认知障碍动物模型的研究进展》)。
脑小血管病公认的动物模型主要包括双侧颈总动脉结扎模型、双侧颈总动脉狭窄模型(BCAS)、卒中易感性自发高血压大鼠模型等(《脑小血管病转化医学研究中国专家共识》)。
作为血管性认知障碍、脑小血管病研究中较为公认的实验动物模型，BCAS模型动物的脑血流量显著降低，引起慢性低灌注损伤，导致血脑屏障通透性增加、白质损伤，进而引起记忆功能障碍、运动功能损伤等。本申请研究结果显示激肽释放酶I或其衍生物给予BCAS模型小鼠后可显著改善受损的运动协调功能、前肢受损肌肉强度、工作记忆、空间学习记忆功能。因此激肽释放酶I或其衍生物 可以用于治疗血管性认知功能障碍、脑小血管病。
此外，卒中后认知功能障碍、脑小血管病导致的认知功能障碍、伴有血管病变的阿尔兹海默病等均为血管性认知功能障碍的重要亚型，在各自临床治疗上，它们会用到相同的对症治疗药物，如针对认知功能障碍，血管性认知功能障碍、卒中后认知功能障碍、脑小血管病的治疗指南或专家共识普遍推荐使用乙酰胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂(美金刚)；针对精神行为症状如抑郁的药物治疗普遍推荐使用抗抑郁药。因此激肽释放酶I或其衍生物可以用于治疗各个亚型的血管性认知功能障碍，如卒中后认知功能障碍、脑小血管病导致的认知功能障碍、伴有血管病变的阿尔兹海默病等。
激肽释放酶-激肽系统(KKS，Kinin-kallikrein system)，参与多种生理和病理过程，如心血管、肾脏和神经系统的功能调节。KKS系统包括激肽释放酶、激肽原、激肽、激肽受体(B1、B2受体)及激肽酶。激肽释放酶，又称激肽原酶或血管舒缓素，是一种丝氨酸蛋白酶，分为两大类：血浆激肽释放酶I(PK)和组织激肽释放酶I(TK)，均发挥十分重要的生理作用。目前认为人组织激肽释放酶至少由15个成员(KLK1-KLK15)组成，其中对组织激肽释放酶I(KLK1)的研究较多，KLK1通过将激肽原转化为激肽，作用于相应受体，发挥一系列生物作用。国内已有两种注射用激肽释放酶I上市，适应症包括微循环障碍性疾病以及轻-中度急性缺血性脑卒中。
血管性认知功能障碍、脑小血管病等与激肽释放酶I已获批适应症有较大不同。在病理机制方面，急性缺血性脑卒中是在各种原因引起的血管壁病变基础上，脑动脉主干或分支动脉管腔狭窄、闭塞或血栓形成，引起脑局部血流减少或供血中断，使脑组织缺血、缺氧性坏死，出现局灶性神经系统症状和体征。VCI是指由脑血管病危险因素(如高血压、糖尿病、高血脂等)、明显(如脑梗死和脑出血等)或不明显的脑血管病(如白质疏松、慢性脑缺血)引起的从轻度认知障碍到痴呆的一大类综合征。脑小血管病的发病过程中神经血管单元(neurovascular unit，NVU)功能异常起重要作用，任何原因引起的NVU结构或功能改变均可导致CSVD，常见机制包括慢性脑缺血和低灌注、内皮功能障碍和血脑屏障破坏、组织间液回流障碍、炎症反应和遗传因素等，不同机制 存在相互作用。(the neurovaseular unit coming of age：a journey through neurovaseular coupling in health and disease)
此外，急性缺血性脑卒中与血管性认知障碍及脑小血管病的诊断、治疗方案均有较大差别。急性缺血性脑卒中最有效的治疗方法是时间窗内给予血管再通治疗，包括静脉溶栓、机械取栓、血管成形术等，救治成功率与发病时间密切相关。药物治疗包括抗血小板、抗凝、降纤、扩容、扩张血管、他汀类药物、神经保护药物等。VCI、PSCI通常会出现认知功能损害和精神行为症状，针对VCI和PSCI认知功能损害的药物有胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂，精神行为症状针对性治疗药物，如抗抑郁药物5-羟色胺再摄取抑制剂等。慢性脑小血管病患者可出现认知障碍、运动障碍、情感障碍和二便障碍等症状，一般建议在明确诊断后进行对症处理。如CSVD导致的认知障碍可选择胆碱酯酶抑制剂、非竞争性N-甲基-D-天冬酰胺受体拮抗剂，抑郁一般选用抗抑郁药物如5-羟色胺再摄取抑制剂等。
本申请研究发现激肽释放酶I或其衍生物可以用于治疗血管性认知功能障碍、卒中后认知功能障碍、脑小血管病等。与激肽释放酶I已获批适应症有较大不同，提供了一种治疗血管性认知功能障碍、卒中后认知功能障碍、脑小血管病的新方法。
附图说明
图1：KLK1及其突变体激活下游CREB蛋白的磷酸化。
图2：Lys-BK激活下游ERK1/2和CREB蛋白的磷酸化。
图3：实验一第13天药物对转棒跌落时间(Rotarod test)的影响。
图4：实验一第26天药物对转棒跌落时间(Rotarod test)的影响。
图5：实验一第20天药物对铁丝跌落时间(Wire hang)的影响。
图6：实验一第28天药物对铁丝跌落时间(Wire hang)的影响。
图7：实验一第29天药物对Y Maze轮流得分的影响。
图8：实验一第44天药物对Y Maze轮流得分的影响。
图9：实验一第48天药物对Morris maze登台潜伏期的影响。
图10：实验二药物对转棒跌落时间(Rotarod test)的影响。
图11：实验二药物对铁丝跌落时间(Wire hang)的影响。
图12：实验二药物对Morris maze登台潜伏期(在III象限活动的路程)的影响。
图13：实验二药物对Morris maze登台潜伏期(在III象限活动的时间)的影响。
具体实施方式
除非特别指明，否则本申请的技术术语或简称具有下列含义：
hKLK1：与天然人组织激肽释放酶I序列一致、未突变的人组织激肽释放酶I；涵盖人KLK1的各种同源物，包括但不限于Genbank登录号为AAA59455.1、NP002248.1、AAA36136.1、AAP35917、AAU12569等所示的KLK1。在具体的实施例中序列如SEQ ID NO：5所示。
KLK1三个糖基化修饰位点N78、N84与N141：分别指KLK1氨基酸序列第78位、84位、141位的天冬酰胺。对应的N糖基化三联子序列基序分别为NMS、NHT、NFS，N代表天冬酰胺，M代表甲硫氨酸，S代表丝氨酸，H代表组氨酸，T代表苏氨酸，F代表苯丙氨酸。
高糖基化hKLK1：未突变的hKLK1，具有高糖基化修饰，即在N78、N84、N141处的糖基化修饰位点均有较多糖修饰。
低糖基化hKLK1：未突变的hKLK1，具有低糖基化修饰，即在N78、N84处的糖基化修饰位点均有较多糖修饰，N141处的糖基化修饰位点无发生糖基化修饰，或仅有少量发生糖基化修饰。重组高糖基化hKLK1、低糖基化hKLK1可以通过常规的纯化方法分离，如疏水层析、阴离子层析、阳离子层析或其组合。申请人意外地发现低糖基化KLK1的活性远高于高糖基化KLK1。
PEG-hKLK1(高糖基化)：聚乙二醇修饰的高糖基化hKLK1，所述高糖基化hKLK1指上述未突变、具有高糖基化修饰的hKLK1。
PEG-hKLK1(低糖基化)：聚乙二醇修饰的低糖基化hKLK1，所述低糖基化hKLK1指上述未突变、具有低糖基化修饰的hKLK1。
hKLK1X：hKLK1突变体。KLK1是具有丰富糖基化修饰的酶，N-糖基化修饰是糖蛋白药物中最受关注的糖基化修饰，糖链通过与新生肽链中特定天冬酰胺的自由-NH 2基连接。N-糖基化修饰位点的三联子序列基序必须为NXS或NXT，其中N表示天冬酰胺，S表示丝氨酸，T表示苏氨酸，X表示除脯氨酸外的其他氨基酸，不满足上述条件的天冬酰胺不发生N-糖基化修饰。hKLK1X1、 hKLK1X2、hKLK1X3、hKLK1X4代表不同突变体，序列分别如SEQ ID NO：1、2、3、4所示。它们对糖基化修饰位点N141进行点突变，将天冬酰胺分别突变成中性极性氨基酸谷氨酰胺(Gln)，酸性氨基酸天冬氨酸(Asp)，碱性氨基酸精氨酸(Arg)，脂肪族氨基酸丙氨酸(Ala)四种不同类型的氨基酸，突变体无NFS的N糖基化基序、仅有两个糖基化修饰位点，从而实现产物更均一、低糖基化hKLK1产量更高的效果，并且令人意外地发现突变后的低糖基化KLK1的活性远高于未突变的低糖基化KLK1。
上述仅用于例举，而非限制本申请的范围。下列方案均能改变原有的NFS序列、使之不构成N糖基化基序，从而无法在N141处发生糖基化修饰，从而实现产物更均一、低糖基化hKLK1产量更高的效果。如突变NFS处的N(即N141)，F、S中0个、1个或2个氨基酸突变为其他任意氨基酸；或NFS处的F(即F142)突变为脯氨酸，N、S中0个、1个或2个氨基酸突变为其他任意氨基酸；或NFS处的S(即S143)突变为除苏氨酸外的任意氨基酸，N、F中0个、1个或2个氨基酸突变为其他任意氨基酸。并且令人意外地发现突变后的低糖基化KLK1的活性远高于未突变的低糖基化KLK1。
PEG-hKLK1X：聚乙二醇修饰的hKLK1突变体。
KLK1衍生物：既包括KLK1全长蛋白，也包括KLK1的部分蛋白；既包括本申请KLK1突变体，也包括在本申请KLK1突变体的基础上进一步突变获得的蛋白、融合蛋白(包括但不限于白蛋白融合、Fc融合等)、各种形式修饰物(包括但不限于聚乙二醇化修饰物)。
聚乙二醇：PEG，通常经环氧乙烷聚合而成，有分支型，直链型和多臂型。一般情况下，分子量低于20,000的被称为PEG，分子量更大的被称为PEO。普通的聚乙二醇两端各有一个羟基，若一端以甲基封闭则得到甲氧基聚乙二醇(mPEG)。
聚乙二醇修饰剂：PEG修饰剂，指带有官能团的聚乙二醇衍生物，是经过活化的聚乙二醇，可用于蛋白质以及多肽药物修饰。本申请所用聚乙二醇修饰剂购自江苏众红生物工程创药研究院有限公司或北京键凯科技股份有限公司。特定分子量的PEG修饰剂实际分子量可以是标示值的90％～110％，如PEG5K分子量可以是4.5kDa～5.5kDa。
实施例所用的PEG5K具体指M-SPA-5K，是分子量约为5kDa的直链单甲氧基聚乙二醇琥珀酰亚胺丙酸酯，结构如式(1)所示，n为105至110的整数，
Figure PCTCN2022144054-appb-000001
实施例所用的PEG10K具体指M-SPA-10K，是分子量约为10kDa的直链单甲氧基聚乙二醇琥珀酰亚胺丙酸酯，结构如式(1)所示，n为220至225的整数。
实施例所用的PEG30K具体指Y-PALD-30K，是分子量约为30kD的分支型聚乙二醇丙醛，结构如式(2)所示，m为335至340的整数，
Figure PCTCN2022144054-appb-000002
实施例所用的PEG40K具体指Y-PALD-40K，是分子量约为40kD的分支型聚乙二醇丙醛，结构如式(2)所示，m为450至455的整数。
适合KLK1的聚乙二醇修饰剂不限于具体实施例使用的上述聚乙二醇修饰剂，还可以尝试其他公知的聚乙二醇修饰剂。
以下实施例所采用的KLK1的聚乙二醇化修饰物，为使用本领域公知的方法进行聚乙二醇修饰并纯化获得聚乙二醇修饰的蛋白。例如：可参考中国专利CN109498815A、中国专利CN107760661A或在先申请CN202111353294.2制备。
实施例1重组人激肽释放酶I(hKLK1)及其突变体(hKLK1X)的体外活性检测
一、基于人工底物的体外活性评价
激肽释放酶I在体内催化低分子激肽原LMWK水解释放赖氨酰缓激肽发挥生物学功能，水解反应涉及精氨酸(Arg)羧基端肽键的断裂。因此，可基于水解人工合成发色底物S-2266(H-D-Val-Leu-Arg-PNA)中Arg与对硝基苯胺间酰胺键断裂生成对硝基苯胺(PNA)，在405nm下检测PNA的生成以评价重组hKLK1及其突变体的体外生物学活性。活性单位IU定义为在37℃、pH8.0条件下，每分钟水解1μmol S-2266为PNA的酶量为1IU。反应体系为200μl 20mM 三羟甲基氨基甲烷缓冲液，10μl供试品，20μl 20mM S-2266底物溶液，置于37℃水浴中准确反应10min，加入20μl 50％醋酸溶液终止反应，基于不同浓度的PNA标准品拟合的标准曲线定量反应体系中PNA的生成量。运用上述方法完成高低糖基化hKLK1、hKLK1突变体的体外生物学活性检测。
结果如下表所示，突变体(hKLK1X1、hKLK1X2、hKLK1X3、hKLK1X4)样品的活性均高于未突变的低糖基化hKLK1样品。
表1
样品名称 比活性IU/mg 相对活性
低糖基化hKLK1 5.1 100％
hKLK1X1 7.0 137.3％
hKLK1X2 7.1 139.2％
hKLK1X3 5.7 111.8％
hKLK1X4 5.7 111.8％
尤瑞克林 4.5 88.2％
二、基于天然底物的体外活性评价
1、酶促反应与液相检测
KLK1在体内通过催化低分子激肽原LMWK水解释放赖氨酰缓激肽发挥生物学功能。本实施例比较了底物(低分子激肽原LMWK)和酶(KLK1或其PEG修饰物)不同比例条件下，酶促反应生成效应分子缓激肽的情况，经反相色谱法对生成的效应分子进行分离、计算产物峰面积，绘制不同底物与酶的比例下缓激肽的生成情况曲线，比较不同供试样品在相同反应条件下的效应分子生成量，通过这种在体外模拟体内作用方式的手段，间接比较不同供试样品的体内作用效果。
供试品中，Kb为猪胰脏提取的KLK1；PEG-Kb是PEG10K修饰的Kb；PEG10K-hKLK1X1、PEG-Kb可按照常规的聚乙二醇修饰药物的方法制备。
按下表对样品进行混合反应(PEG修饰样品中，以其中被修饰的活性物质的摩尔质量进行换算)，混合样品置于37℃恒温器中，孵育15min，精确计时，按照体积比10：1加入50％醋酸溶液终止反应。
表2
体系(底物：酶 底物μl 酶μl 50％醋酸μl 体系总体积μl
mol/mol)        
1：1 10 10 2 22
5：1 50 10 6 66
10：1 50 5 5.5 60.5
15：1 60 4 6.4 70.4
20：1 60 3 6.3 69.3
终止反应后的样品，置于台式离心机，12000rpm离心5min，取上清。Waters ACQUITY UPLC H-Class检测，流动相A为0.1％TFA-H 2O，流动相B为0.1％TFA-ACN，检测波长214nm，柱温30℃，上样量均为10μl，流速0.2ml/min，运行时间35min，运行梯度如下：
表3
Figure PCTCN2022144054-appb-000003
2、结果
根据酶促反应混合物的UPLC色谱图结果，对反应混合物色谱图中保留时间为13±0.5min(缓激肽出峰位置)的色谱峰进行积分并求和，并与单位质量(1mg/ml，10μl上样量)的缓激肽峰面积比较，计算酶促反应生成的缓激肽的浓度，并根据表2中反应总体系计算缓激肽生成的量，最终折算成在每个摩尔比条件下每毫克酶对应的缓激肽生成量(μg/mg)，结果如表4所展示。
表4
Figure PCTCN2022144054-appb-000004
Figure PCTCN2022144054-appb-000005
基于天然底物的检测结果显示，整体趋势上，修饰后蛋白经酶促反应生成的缓激肽低于修饰前样品，符合PEG修饰蛋白的一贯特性，经过PEG修饰后的蛋白相对于原蛋白能更温和的维持酶促过程，达到效应分子的持续释放；同时KLK1类药物的起效是基于体内KKS系统的调节作用，这种调节包含了效应分子的释放与清除，显然，温和而持续的酶促过程可以有效地减少KKS系统的清除机制，使得PEG修饰KLK1能更稳定、有效的发挥生物学作用。
实施例2 KLK1对B2受体下游信号的激活作用
收集处于对数生长期的CHO-K1细胞(含B2受体)，用血球计数板进行活细胞计数。用培养基将活细胞悬液调整至3×10 6cells/mL接种于6孔细胞培养板，最终细胞浓度为3×10 5个/孔。细胞于37.0℃，5.0％CO 2的CO 2培养箱培养24～48h。待每孔细胞密度长到90％以上时，用PBS洗涤2遍。提前用F12培养基配制好样品(KLK1和LMWK按质量比1：10混和，反应体系为F12培养基，反应15min)，反应结束后每孔加入1ml样品，KLK1终浓度为0.1μM，对照组则为F12培养基。加药完成后，将细胞培养板置于37.0℃、5.0％CO 2培养箱中孵育0.5h。孵育结束后，将6孔板从培养箱取出，用PBS洗涤2遍，最后1遍将PBS吸尽。将配制好的蛋白裂解液(RIPA、PMSF、磷酸酶抑制剂)按80μL/孔加入，用刮棒来回研磨孔底以加速细胞裂解，研磨时间1min。将细胞碎片与裂解液转移至离心管中，12000rpm，10min，4℃离心。小心吸取蛋白上清，将蛋白上清与5×Loading Buffer以4:1体积比混匀，后于100℃金属浴煮沸10min；样品于室温冷却后，短时间置于4℃保存，长时间-20℃保存。将等量的蛋白样品和预染marker上样到10％SDS-PAGE中进行跑胶、转膜、封闭、抗体孵育、发光鉴定等操作。
环磷腺苷效应元件结合蛋白(cAMP-response element binding protein,CREB)是B2R激活后下游直接作用的信号分子，CREB的激活可以防止缺血后的炎症反应与神经元损伤的发生，故其表达的高低可一定程度反应受试物激活B2R的能力，同时从侧面反应神经保护效应发挥的意义。
基于B2-NFAT-CHO-K1细胞株证明了KLK1可催化低分子激肽原LMWK生成活性分子与B2受体结合后激活下游信号CREB蛋白的活化，如图1所示，可以激活下游CREB蛋白的磷酸化，与该药物作用机制存在一致性，通过判断下游CREB蛋白的磷酸化水平来定性评价样品的体外生物学活性。结果显示，hKLK1X1、hKLK1X2、hKLK1X3、hKLK1X4、低糖基化hKLK1、高糖基化hKLK1与LMWK反应后均显示出激活下游通路的活性。
实施例3赖氨酰缓激肽Lys-BK对B2受体下游信号的激活作用
收集处于对数生长期的CHO-K1(含B2受体)细胞，用血球计数板进行活细胞计数。用培养基将活细胞悬液调整至3×10 6cells/mL接种于6孔细胞培养板，最终细胞浓度为3×10 5个/孔。细胞于37.0℃，5.0％CO 2的CO 2培养箱培养24～48h。待每孔细胞密度长到90％以上时，用PBS洗涤2遍。提前用F12培养基配制好样品(Lys-BK)，每孔加入1ml样品，Lys-BK终浓度为1μM，对照组则为F12培养基。加药完成后，将细胞培养板置于37.0℃、5.0％CO 2培养箱中孵育0.5h。孵育结束后，将6孔板从培养箱取出，用PBS洗涤2遍，最后1遍将PBS吸尽。将配制好的蛋白裂解液(RIPA、PMSF、磷酸酶抑制剂)按80μL/孔加入，用刮棒来回研磨孔底以加速细胞裂解，研磨时间1min。将细胞碎片与裂解液转移至离心管中，12000rpm，10min，4℃离心。小心吸取蛋白上清，将蛋白上清与5×Loading Buffer以4:1体积比混匀，后于100℃金属浴煮沸10min；样品于室温冷却后，短时间置于4℃保存，长时间-20℃保存。将等量的蛋白样品和预染marker上样到10％SDS-PAGE中进行跑胶、转膜、封闭、抗体孵育、发光鉴定等操作。
胞外信号调节激酶(extracellular regulated protein kinases 1/2,ERK1/2)、环磷腺苷效应元件结合蛋白(cAMP-response element binding protein,CREB)都是B2R激活后下游直接作用的信号分子，CREB与ERK1/2的激活可以防止缺血后的炎症反应与神经元损伤的发生，故其表达的高低可一定程度反应受试物激活B2R的能力，同时从侧面反应神经保护效应发挥的意义。
基于B2-NFAT-CHO-K1细胞株证明了赖氨酰缓激肽Lys-BK与B2受体结合后激活下游信号CREB、p-ERK1/2蛋白的活化，如图2所示，Lys-BK可以激活下游CREB、p-ERK1/2蛋白的磷酸化。
实施例4 KLK1-PEG在小鼠BCAS模型上的药效作用
实验一
一、分组与实验设计
采用双侧颈总动脉弹簧缩束法制备小鼠双侧颈总动脉狭窄(Bilateral common carotid artery stenosis,BCAS)脑小血管缺血损伤模型。
BCAS脑小血管损伤模型的制备：采用BCAS脑小血管缺血损伤模型，实现小鼠持续性前脑慢性缺血。小鼠使用异氟烷麻醉，首先将小鼠放入麻醉机的诱导盒中诱导麻醉，然后将小鼠仰卧保定并连接呼吸面罩，备皮、消毒皮肤，颈部正中切开，分离双侧颈总动脉，用定制弹簧(弹簧材料：进口钢琴丝，尺寸：线径：0.08mm，内径：0.18mm，节距：约0.5mm，总长：2.5mm)缩束双侧颈总动脉，阻断大部分血流，造成脑部血供降低，全脑持续性慢性缺血。缝合颈部皮肤，消毒，放回笼中饲养。
试验共设3组，即假手术组(SHAM组)、模型对照组(BCAS组)、候选药物组(KLK1-PEG组)。
于BCAS术后第3天开始分组给药，每周给药一次，共给药7次。KLK1-PEG组(80μg/kg，所述KLK1是hKLK1X1，PEG是SPA10K)、BCAS组及SHAM组每周尾静脉注射等体积生理盐水1次。
采用转棒实验(Rotarod test)于术后第13天、26天检测小鼠运动协调功能。将小鼠按旋转相反的方向放置于小鼠转棒(30mm直径×60mm长)上，启动转棒，以20rpm/min均匀加速至转速为40rpm/min后维持。记录小鼠在棒时间(即小鼠跌落时间)为指标。每只小鼠需要检测3～5次，每次间隔为20～30min。取均值作为小鼠转棒跌落时间，转棒跌落时间越长表明运动协调功能越好。
采用挂绳实验(Wire hang)于术后第20天、28天检测小鼠前肢肌肉强度受损情况。将小鼠前肢悬挂于钢丝(1.5mm直径×60cm长)上，记录小鼠在钢丝上悬挂的时间(即小鼠跌落时间)为指标。需要检测3～5次，每次间隔为20～30min，取均值作为小鼠铁丝跌落时间。铁丝跌落时间越长表明肌肉受损越轻微。
采用Y迷宫实验(Y Maze)于术后第29天、44天检测小鼠工作记忆受损情况。Y迷宫是由三个完全相同的臂互相成120°组成的“Y形”实验设备，每个臂长30cm宽8cm高15cm，利用了啮齿类动物对新环境探索的天性，用于 学习记忆等实验研究。将小鼠放置于Y迷宫中，让小鼠自由活动。记录7min内，小鼠进入各个臂(四只爪子都进入臂)的顺序。所有进臂次数定义为进臂次数，连续进入所有三个臂的次数被定义为轮流次数，以轮流得分为Y Maze评价指标。轮流得分＝[轮流次数/(进臂次数-2)]*100。在试验过程中，淘汰进入臂少于8次的动物(因其的数据不能反映精确的变化)。轮流得分越高表明工作记忆受损越轻微。
采用水迷宫实验(Morris maze)于术后第48天检测小鼠空间学习记忆受损情况。水迷宫水温保持(23±2)℃，池壁上标有东南西北四个入水点，将水池等分为四个象限(E、S、W、N)，迷宫上方安置带有显示系统的摄像机，用以同步记录小鼠运动轨迹。训练期间迷宫外参照物保持不变，以供大鼠定位平台。以登台潜伏期(小鼠从入水至找到平台的时间)作为检测指标，登台潜伏期越短空间学习记忆受损越轻微。
二、结果
BCAS组与SHAM组比较，在转棒实验、挂绳实验、Y迷宫实验、水迷宫实验的结果上均有显著性统计学差异，表明BCAS造模导致动物的运动协调、前肢肌肉强度、工作记忆和空间学习记忆障碍。KLK1-PEG组与BCAS造模组比较，上述各项检测均有改善。BCAS模型是血管性认知障碍、脑小血管病研究中较为公认的实验动物模型，上述实验结果说明KLK1或其衍生物可用于治疗脑小血管病、血管性认知障碍。
结果详见附图3-9、表5～表8。
表5 静脉注射给药对Rotarod test转棒跌落时间的影响
Figure PCTCN2022144054-appb-000006
Figure PCTCN2022144054-appb-000007
表6 静脉注射给药对Wire hang铁丝跌落时间的影响
Figure PCTCN2022144054-appb-000008
***P<0.001，**P<0.01，与SHAM组相比。
表7 静脉注射给药对Y Maze轮流得分的影响
Figure PCTCN2022144054-appb-000009
***P<0.001，**P<0.01，*P<0.05，与SHAM组相比。
表8 静脉注射给药对Morris maze登台潜伏期的影响
Figure PCTCN2022144054-appb-000010
Figure PCTCN2022144054-appb-000011
实验二
一、分组与实验设计
采用双侧颈总动脉弹簧缩束法制备小鼠双侧颈总动脉狭窄(Bilateral common carotid artery stenosis,BCAS)脑小血管缺血损伤模型。
BCAS脑小血管损伤模型的制备同实验一。
试验共设3组，即模型组(BCAS组)、KLK1组、KLK1-PEG组(所述KLK1是hKLK1X1，PEG是SPA10K)。
于BCAS术后72小时开始分组给药。KLK1组(0.5mg/kg)每周静脉注射给药5次、KLK1-PEG组(0.1mg/kg)每周肌肉注射给药1次，共给药8周。
采用转棒实验(Rotarod test)于给药8周后检测小鼠运动协调功能，评价方法同实验一。采用挂绳实验(Wire hang)于给药8周后检测小鼠前肢肌肉强度受损情况，评价方法同实验一。采用水迷宫实验(Morris maze)于给药8周后检测小鼠空间学习记忆受损情况，将水面分为4个象限，并将平台放置在III象限的中心；在测试阶段去除水中的平台，纪录动物在不同象限活动的时间、路程；动物在III象限活动的时间和路程占总时间和总路程的比例反应了动物的记忆能力，比例越高说明空间学习记忆受损越轻微。
二、结果
KLK1组、KLK1-PEG组与模型组比较在转棒实验、挂绳实验、水迷宫实验的结果上均有显著性统计学差异，KLK1或KLK1-PEG能显著改善BCAS造模导致动物的运动协调、前肢肌肉强度、空间学习记忆障碍，可用于治疗脑小血管病、血管性认知障碍。结果详见附图10～13、表9～表11。
表9 静脉注射给药对Rotarod test转棒跌落时间的影响
组别 样本(只) 转棒跌落时间(s)均值±标准误
BCAS组 11 41.15±3.94
KLK1组 11 70.06±9.05**
KLK1-PEG组 11 83.66±10.94**
**表示与模型组相比P<0.01。
表10 给药对Wire hang铁丝跌落时间的影响
组别 样本(只) 铁丝跌落时间(s)均值±标准误
BCAS组 12 10.83±1.76
KLK1组 12 16.50±1.46*
KLK1-PEG组 10 21.80±4.33*
*表示与模型组相比P<0.05。
表11 静脉注射给药对Morris maze登台潜伏期的影响
组别 样本(只) III象限路程(％)均值±标准误 III象限时间(％)均值±标准误
BCAS组 11 24.22±2.36 24.39±2.54
KLK1组 16 32.08±1.82* 32.24±1.98*
KLK1-PEG组 15 29.86±2.51* 28.08±2.40*
*表示与模型组相比P<0.05。
实施例采用了作为血管性认知障碍、脑小血管病研究中较为公认的实验动物模型，即BCAS模型，BCAS模型小鼠给予KLK1或其聚乙二醇修饰物后均可改善受损的运动协调功能、前肢受损肌肉强度、工作记忆、空间学习记忆功能，可以用于治疗血管性认知功能障碍、卒中后认知功能障碍、脑小血管病。
除上述受试药外，BCAS模型小鼠给予其他较好地保留KLK1催化低分子激肽原LMWK水解释放赖氨酰缓激肽发挥生物学功能的KLK1衍生物，如KLK1其他突变体(hKLK1X2、hKLK1X3、hKLK1X4等)、部分蛋白、融合蛋白、其他PEG修饰物、其他形式修饰物时，均能观察到类似作用，即改善BCAS模型 小鼠受损的运动协调功能、前肢受损肌肉强度、工作记忆、空间学习记忆功能，可以用于治疗血管性认知功能障碍、卒中后认知功能障碍、脑小血管病。

Claims (16)

  1. 激肽释放酶I或其衍生物在制备治疗或改善血管性认知功能障碍的药物中的应用。
  2. 一种用于治疗或改善血管性认知功能障碍的药物组合物，包含激肽释放酶I或其衍生物。
  3. 一种治疗或改善血管性认知功能障碍的方法，即给予患者激肽释放酶I或其衍生物。
  4. 激肽释放酶I或其衍生物在制备治疗或改善卒中后认知功能障碍的药物中的应用。
  5. 一种用于治疗或改善卒中后认知功能障碍的药物组合物，包含激肽释放酶I或其衍生物。
  6. 一种治疗或改善卒中后认知功能障碍的方法，即给予患者激肽释放酶I或其衍生物。
  7. 激肽释放酶I或其衍生物在制备治疗或改善伴有血管病变的阿尔兹海默病的药物中的应用。
  8. 一种用于治疗或改善伴有血管病变的阿尔兹海默病的药物组合物，包含激肽释放酶I或其衍生物。
  9. 一种治疗或改善伴有血管病变的阿尔兹海默病的方法，即给予患者激肽释放酶I或其衍生物。
  10. 激肽释放酶I或其衍生物在制备治疗或改善脑小血管病或脑小血管病导致的认知功能障碍的药物中的应用。
  11. 一种用于治疗或改善脑小血管病或脑小血管病导致的认知功能障碍的药物组合物，包含激肽释放酶I或其衍生物。
  12. 一种治疗或改善脑小血管病或脑小血管病导致的认知功能障碍的方法，即给予患者激肽释放酶I或其衍生物。
  13. 如权利要求1至12任一项所述的激肽释放酶I为天然人组织激肽释放酶I，突变或未突变的重组人组织激肽释放酶I。
  14. 如权利要求1至12任一项所述的激肽释放酶I衍生物为激肽释放酶I的全长蛋白、部分蛋白或者突变体、融合蛋白、各种形式修饰物。
  15. 如权利要求1至12任一项的激肽释放酶I衍生物为激肽释放酶I的聚 乙二醇化修饰物。
  16. 如权利要求15所述的激肽释放酶I的聚乙二醇化修饰物为以M-SPA-5K、M-SPA-10K、Y-PALD-30K、Y-PALD-40K修饰的激肽释放酶I。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101134105A (zh) * 2007-07-02 2008-03-05 广东天普生化医药股份有限公司 用于治疗和/或预防脑梗塞的含有重组人胰激肽原酶的药物组合物
CN101530611A (zh) * 2009-04-09 2009-09-16 中山大学附属第一医院 I型激肽释放酶在制备防治脑损伤后远隔部位继发性损害的药物中的新用途
CN101801406A (zh) * 2007-07-20 2010-08-11 基因塞斯投资有限公司 用于治疗淀粉样蛋白相关疾病的组织激肽释放酶
CN104073482A (zh) * 2013-12-30 2014-10-01 江苏众红生物工程创药研究院有限公司 聚乙二醇化的组织激肽释放酶及其制备方法和应用
CN107760661A (zh) 2016-08-18 2018-03-06 江苏众红生物工程创药研究院有限公司 药用激肽原酶的peg修饰物及其制备方法和应用
CN110446501A (zh) * 2017-03-09 2019-11-12 代阿麦迪卡股份有限公司 组织激肽释放酶1的剂型

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101134105A (zh) * 2007-07-02 2008-03-05 广东天普生化医药股份有限公司 用于治疗和/或预防脑梗塞的含有重组人胰激肽原酶的药物组合物
CN101801406A (zh) * 2007-07-20 2010-08-11 基因塞斯投资有限公司 用于治疗淀粉样蛋白相关疾病的组织激肽释放酶
CN101530611A (zh) * 2009-04-09 2009-09-16 中山大学附属第一医院 I型激肽释放酶在制备防治脑损伤后远隔部位继发性损害的药物中的新用途
CN104073482A (zh) * 2013-12-30 2014-10-01 江苏众红生物工程创药研究院有限公司 聚乙二醇化的组织激肽释放酶及其制备方法和应用
CN109498815A (zh) 2013-12-30 2019-03-22 江苏众红生物工程创药研究院有限公司 重组人激肽释放酶的化学修饰物及其应用
CN107760661A (zh) 2016-08-18 2018-03-06 江苏众红生物工程创药研究院有限公司 药用激肽原酶的peg修饰物及其制备方法和应用
CN110446501A (zh) * 2017-03-09 2019-11-12 代阿麦迪卡股份有限公司 组织激肽释放酶1的剂型

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. AAA36136.1
WANG, FENGSHAN ET AL.: "Physiological, Pharmacological Effects and Clinical Applications of Kallikrein", BIOCHEMICAL RESEARCH, 31 December 1997 (1997-12-31), pages 522 - 527, XP009548373 *

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