Use of quinine or its derivatives in the treatment of VCI, PSCI or CSVD

Abstract

Use of kinine or its derivatives, particularly long-acting kinine or its derivatives, in the treatment of VCI, PSCI, or CSVD. VCI and PSCI often show cognitive dysfunction and psychiatric and behavioral symptoms, and cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists are used for the cognitive dysfunction, and antidepressants are used symptomatically for the psychiatric and behavioral symptoms. Chronic CSVD patients show symptoms such as cognitive impairment, motor disorder, emotional disorder, and defecation disorder, and cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists are used for the cognitive dysfunction, and antidepressants are generally used for depression. The present invention has discovered through research that kinine or its derivatives, particularly long-acting kinine or its derivatives, can be used to treat VCI, PSCI, CSVD, etc., providing a new method for treating these diseases.

Description

[Technical field]

[0001] The present invention relates to the use of kinin or a derivative thereof, particularly long-acting kinin or a derivative thereof, in the biomedicine field 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 cerebrovascular disease (e.g., cerebral infarction and cerebral hemorrhage, etc.). It is a series of syndromes ranging from mild cognitive impairment to dementia caused by non-cancerous cerebrovascular diseases (e.g., white matter sparing and chronic cerebral ischemia) (2019 Chinese Guidelines for the Treatment of Vascular Cognitive Disorder, Chinese Guiding Standards for the Treatment of Vascular Cognitive Disorder in the Chinese Guiding Standards for the Prevention and Treatment of Stroke (2021 Edition)).

[0003] Patients with vascular cognitive dysfunction have cognitive impairment and mental 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 areas, and memory is also significantly impaired. Medications for cognitive impairment in vascular cognitive dysfunction include cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists. Mental and behavioral symptoms of vascular cognitive dysfunction 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 Disorders). The main subtypes of vascular cognitive impairment include post-stroke cognitive impairment (PSCI), cognitive impairment due to cerebral small vessel disease (CSVD), and Alzheimer's disease (AD) accompanied by 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 post-stroke cognitive impairment, vascular cognitive impairment, and Alzheimer's disease have some commonality in neuropathological and neurobiochemical mechanisms, China's "Post-stroke Cognitive Impairment Management Expert Consensus 2021" recommends that in treating post-stroke cognitive impairment, acetylcholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists (memantine) are generally used for cognitive impairment, and antidepressants are generally used for post-stroke depression, based on research and evidence from vascular cognitive impairment and Alzheimer's disease. 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 notes that there is a significant lack of high-quality data from randomized controlled trials on post-stroke cognitive impairment.

[0005] Cerebral small vessel disease 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, resulting in acute cerebral It is classified into small vessel disease and chronic cerebral small vessel disease. Acute cerebral small vessel disease may cause ischemic stroke, and it is currently recommended to treat it with reference to the prevention and treatment of acute ischemic stroke (e.g., blood pressure reduction, thrombolysis, antiplatelet, lipid reduction, etc.). Patients with chronic cerebral small vessel disease show symptoms such as cognitive impairment, motor disorder, emotional disorder, and defecation disorder, and after a clear diagnosis, symptomatic treatment is generally recommended. Cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists are used for cognitive impairment caused by cerebral small vessel disease, 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 a use of quinine 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 impairment, the composition comprising kinin or a derivative thereof.

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

[0015] The present invention also provides a method for treating or ameliorating cerebral small vessel disease. is a method of administering the derivative to a patient. The kinins include bradykinin or lysylbradykinin. The quinine or derivative thereof is a long-acting quinine or derivative thereof.

[0016] Peptide drugs generally have the disadvantages of poor stability in the body and short half-life, and often require long-term and frequent administration, especially as a treatment for chronic diseases. The decomposition of lysylbradykinin / bradykinin in the body is mainly involved in aminopeptidase, carboxypeptidase and angiotensin ACEII, and the dynamic balance of bradykinin in the body is maintained by the kinin releasing enzyme system and the decomposing enzyme system. Lysylbradykinin and bradykinin are more unstable than ordinary peptides, and their half-life is reported to be only a few seconds. Exogenously administered kinin is quickly inactivated by hydrolysis by kinase in the body.

[0017] Currently, two main methods are used to improve the stability of peptide drugs and extend their half-life in vivo to make them long-acting. One is to modify the peptide molecule, for example, by amino acid substitution, cyclization, PEG modification, fusion with long-acting fragments (fusion with Fc fragments, fusion with human serum albumin), addition of fatty acids, etc. The other is to make peptide drugs long-acting by formulation.

[0018] In the specific embodiment of the present invention, two methods are used to extend the half-life of kinin: modifying kinin with polyethylene glycol and optimizing the amino acid sequence of kinin.

[0019] For polyethylene glycol modification, the PEG modifiers used in the specific embodiments of the present invention include SPA, SCM, and SS modifiers, which may be linear or branched.

[0020] Preferably, the PEG modifier is a linear PEG modifier. Preferably, the molecular weight of the PEG modifier is from 2 KD to 20 KD.

[0021] In the specific embodiment of the present invention, cysteine ​​(Cys) is introduced into any position in the amino acid sequence of wild-type lysylbradykinin or bradykinin. Preferably, Lys at position 1 of the amino acid sequence of wild-type lysylbradykinin is mutated to Cys, that is, the amino acid sequence is made to be CRPPGFSPFR.

[0022] In a specific embodiment of the present invention, any number and any type of amino acids are inserted between the 1st and 2nd amino acid residues of the N-terminus of wild-type lysylbradykinin, i.e., the amino acid sequence is KXRPPGFSPFR (X represents any number and any type of amino acid). Or, any number and any type of amino acids are inserted before the 1st amino acid residue of the N-terminus of wild-type bradykinin, i.e., the amino acid sequence is XRPPGFSPFR (X represents any number and any type of amino acid). Experimental results show that the activity is better after the N-terminus is extended. Preferably, 1, 2, or 3 amino acids are inserted. Preferably, 1 or 2 arginines (Arg) are inserted.

[0023] In a specific embodiment of the present invention, a lysylbradykinin or bradykinin derivative (e.g., m14, m12) was prepared by linking two or more lysylbradykinin units, two or more bradykinin units, or a lysylbradykinin unit and a bradykinin unit. The lysylbradykinin or bradykinin unit may be a wild-type lysylbradykinin or bradykinin, or may be a mutant obtained by substituting, inserting, or deleting a part of amino acids based on the wild-type lysylbradykinin or bradykinin.

[0024] In a specific embodiment of the present invention, Phe at position 6 of wild-type lysylbradykinin or Phe at position 5 of wild-type bradykinin is mutated to other amino acids, such as the unnatural amino acids Igl (α-2-indolylglycine) and THI (β-2-thienylalanine). The site-mutated LBK or BK is less likely to be recognized by enzymes in vivo, and therefore its half-life in vivo is effectively extended.

[0025] Preferably, the amino acid sequence of the lysylbradykinin or bradykinin derivative of the present invention is the sequence shown in SEQ ID NO:14.

[0026] The polyethylene glycol modification method and the amino acid sequence optimization method used in the specific examples of the present invention are merely illustrative of the fact that the half-life of kinin can be extended by these methods, and are not intended to limit the specific embodiment of extending the half-life of kinin. Apart from the methods exemplified in the present invention, those skilled in the art can modify peptides to act for a long time using other polyethylene glycol modification methods, other amino acid sequence optimization methods, or other modification methods for making peptide drugs act for a long time (e.g., fusion with Fc fragments, fusion with human serum albumin, addition of fatty acids, formulations, as described above).

[0027] The kinin or derivatives thereof may be administered alone or in combination with other drugs, such as cholinesterase inhibitors and non-competitive N-methyl-D-aspartate receptor antagonists.

[0028] 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, etc.

[0029] 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).

[0030] 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).

[0031] 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.

[0032] The research results of the present invention show that administration of kinin or its derivatives to BCAS model mice significantly improved the animals' impaired coordinated motor function, the strength of the injured muscles of the forelimbs, working memory, spatial learning and memory function and depressive behavior. Therefore, kinin or its derivatives can be used to treat vascular cognitive dysfunction and cerebral small vessel disease.

[0033] In addition, cognitive impairment after stroke, cognitive impairment due to cerebral small vessel disease, and Alzheimer's disease accompanied by vascular lesions are also considered. Alzheimer's disease and Alzheimer's disease are all important subtypes of vascular cognitive impairment, and their clinical treatments use 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, kinin 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.

[0034] 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 (KLK), 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 (PK) and tissue kallikrein (TK), both of which exert very important physiological functions. Currently, human tissue kallikrein is thought to be composed of at least 15 members (KLK1-KLK15), of which tissue kallikrein 1 (KLK1) has been relatively well studied. KLK1 converts kininogen to kinin and acts on the corresponding receptor to exert a series of biological functions. Currently, the more clearly studied kinins are lysylbradykinin (decapeptide) and bradykinin (nonapeptide), the former of which is cleaved to bradykinin by aminopeptidase in vivo, and both have a wide range of physiological functions, including blood pressure regulation and inflammatory responses. On the other hand, lysylbradykinin and bradykinin have very short half-lives in the body, ranging from a few seconds to a few minutes, which greatly limits their druggability. As of now, no kinins are commercially available as pharmaceuticals. Two types of injectable kallikrein 1 are available in China, and their indications include microcirculatory disorders and mild to moderate acute thrombotic stroke.

[0035] 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).

[0036] 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).

[0037] 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 time of onset. Drug treatment includes the use of antiplatelet, anticoagulant, fibrolytic, volume expanding, vasodilator drugs, statins, neuroprotective drugs, etc. Vascular cognitive impairment Intellectual disability often manifests as cognitive impairment and psychiatric and behavioral symptoms. Drugs for cognitive impairment in vascular cognitive impairment and post-stroke cognitive impairment include cholinesterase inhibitors and noncompetitive N-methyl-D-aspartate receptor antagonists, and symptomatic treatments for psychiatric and behavioral symptoms include selective serotonin reuptake inhibitors, which are antidepressants. Patients with chronic cerebral small vessel disease show symptoms such as cognitive impairment, motor disorders, emotional disorders, and bowel disorders, 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 cerebral small vessel disease, and antidepressants such as serotonin reuptake inhibitors are generally used for depression.

[0038] Through research, the present invention reveals that kinin or a derivative thereof can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, cerebral small vessel disease, etc., and provides a new method for the treatment of vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease. [Brief description of the drawings]

[0039] [Figure 1a] Figure 1 shows that Lys-BK and its mutants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. Among the five signals, β-actin is the internal control, p- represents the phosphorylation of the corresponding protein, control is the buffer blank control, and Lys-BK-M06-EH is the sample after mixing and incubating m06 with serum. [Figure 1b] Figure 1 shows that Lys-BK and its mutants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. Among the five signals, β-actin is the internal control, p- represents the phosphorylation of the corresponding protein, control is the buffer blank control, and Lys-BK-M06-EH is the sample after mixing and incubating m06 with serum. [Figure 1c] Figure 1 shows that Lys-BK and its mutants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. Among the five signals, β-actin is the internal control, p- represents the phosphorylation of the corresponding protein, control is the buffer blank control, and Lys-BK-M06-EH is the sample after mixing and incubating m06 with serum. [Figure 1d] Figure 1 shows that Lys-BK and its mutants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. Among the five signals, β-actin is the internal control, p- represents the phosphorylation of the corresponding protein, control is the buffer blank control, and Lys-BK-M06-EH is the sample after mixing and incubating m06 with serum. [Figure 2a]FIG. 2 shows the results of measuring the affinity of PEG conjugates of Lys-BK and its mutants for the B2 receptor (by reporter gene assay). [Figure 2b] FIG. 2 shows the results of measuring the affinity of PEG conjugates of Lys-BK and its mutants for the B2 receptor (by reporter gene assay). [Diagram 3] Figure 3 shows that PEG conjugates of Lys-BK and its mutants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. Among the five signals, β-actin is the internal control, p- represents the phosphorylation of the corresponding protein, and control is the buffer blank control. [Figure 4] FIG. 4 shows the effect of drugs on the latency to fall in the Rotarod test on the 13th day. [Diagram 5] FIG. 5 shows the effect of drugs on the latency to fall in the Rotarod test on the 26th day. [Figure 6] FIG. 6 shows the effect of drugs on the latency to fall in the wire hang test on the 20th day. [Figure 7] FIG. 7 shows the effect of drugs on the latency to fall in the wire hang test on the 28th day. [Figure 8] FIG. 8 shows the effect of drugs on alternation scores in the Y-maze test on day 29. [Figure 9] FIG. 9 shows the effect of drugs on alternation scores in the Y-maze test on day 44. [Figure 10] FIG. 10 shows the effect of drugs on the latency to reach the platform in the Morris water maze test on day 48. [Figure 11] FIG. 11 shows the effect of drugs on sugar water preference test (SPT) on the 57th day. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Unless otherwise specified, technical terms or abbreviations in the present invention have the following meanings: Lys-BK: wild-type lysylbradykinin, which is identical to the amino acid sequence of natural human lysylbradykinin, and whose amino acid sequence is KRPPGFSPFR.

[0041] BK: wild-type bradykinin, which is identical to the amino acid sequence of native human bradykinin, and whose amino acid sequence is RPPGFSPFR.

[0042] Mutant: A mutant obtained by substituting, inserting or deleting some amino acids on the basis of wild-type lysylbradykinin or bradykinin.

[0043] Derivatives: Derivatives include not only the various mutants exemplified in the examples of the present invention, but also peptides obtained by further modification based on the Lys-BK / BK mutants of the present invention, fusion proteins (including but not limited to albumin fusion, Fc fusion, etc.), and various types of modified products.

[0044] Polyethylene glycol: abbreviated as PEG, is usually formed by polymerization of ethylene oxide and is available in branched, linear and multi-armed forms. Normal polyethylene glycol has hydroxyl groups at both ends, and blocking one end with a methyl group gives methoxypolyethylene glycol (mPEG).

[0045] 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 peptide 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% to 110% of the indicated value. For example, the molecular weight of PEG5K may be 4.5 kDa to 5.5 kDa.

[0046] M-SPA-5K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of approximately 5 kDa, structural formula of which is shown in formula (1), where n is an integer of 98-120.

[0047] [ka]

[0048] M-SPA-2K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of approximately 2 kDa, structural formula of which is shown in formula (1), where n is an integer of 37-45.

[0049] M-SPA-10K: A linear monomethoxy polyethylene glycol succinimidyl propionate having a molecular weight of approximately 10 kDa, and its structural formula is as shown in formula (1), where n is an integer of 200 to 245.

[0050] M-SPA-20K: A linear monomethoxy polyethylene glycol succinimidyl propionate having a molecular weight of about 20 kDa, the structural formula of which is shown in formula (1), where n is an integer of 405-495.

[0051] M-SCM-5K: linear monomethoxy polyethylene glycol succinimidyl carboxymethylate with a molecular weight of approximately 5 kDa, structural formula is as shown in formula (2), where n is an integer of 99-120.

[0052] [ka]

[0053] M-SS-5K: linear monomethoxy polyethylene glycol succinimidyl succinate with a molecular weight of approximately 5 kDa, structural formula of which is shown in formula (3), where n is an integer of 98-119.

[0054] [ka]

[0055] M-YNHS-10K: Branched monomethoxy polyethylene glycol succinimidyl carboxymethylate having a molecular weight of about 10 kDa, the structural formula of which is shown in formula (4), where n is an integer of 98 to 120.

[0056] [ka] EXAMPLES

[0057] Example 1 Design and synthesis of mutants of lysylbradykinin / bradykinin Based on the sequence of lysylbradykinin / bradykinin and its possible degradation pathway in vivo, a series of mutants were designed (Table 1-1). These are mainly mutants in which amino acids in the lysylbradykinin / bradykinin sequence have been replaced with structurally similar natural or non-natural amino acids (m01-m04, m07, and m02 are BK mutants), partially truncated or extended peptide mutants (m05, m06, m08-m11) designed to identify the minimum active unit that exerts the biological activity / function of lysylbradykinin / bradykinin, mutants (m13, m15, and m16) in which cysteine ​​has been introduced to further enhance the antioxidant ability of the mutants, mutants in which lysylbradykinin / bradykinin units have been linked to further improve the stability of the peptide (m12 is a mutant in which Lys-BK and BK are linked, and m14 is a mutant in which two Lys-BKs are linked), and mutants m15 and m16 designed based on peptide m13 to compare the effect of N-terminal extension on the drug efficacy. The purity of all peptides is >95%.

[0058] [Table 1]

[0059] Example 2 Stability studies of lysylbradykinin / bradykinin and its mutants The stability of lysylbradykinin / bradykinin was evaluated in an in vitro serum system. 2mg / ml peptide and SD rat serum were mixed at a volume ratio of 85:15 and incubated at 37℃ for different periods of time: 30, 60, 90, 120, and 180 min. The assay was terminated by adding 3% trichloroacetic acid (50% of the volume of the above peptide-serum mixture), which effectively eliminated the interference of the substrate in serum. Furthermore, the UPLC-RP-UV 214nm The stability of peptide samples was evaluated by monitoring their degradation in serum systems based on the RP method. RP analysis method: The chromatography column was Protein BEH C4 2.1x150mm, 1.7μm, the column temperature was 30℃, mobile phase A: 0.1% TFA / H2O, mobile phase B: 0.1% TFA / ACN, gradient: 0-5min, 5%B; 5-10min, 5-40%B; 10-30min, 40-60%B; 30-32min, 60-100%B; 32-35min, 5%B.

[0060] [Table 2]

[0061] The results in Table 2-1 show that the mutants of several peptides designed in the present invention have better stability in serum compared to wild-type lysylbradykinin, suggesting that the mutants have advantages in terms of in vivo stability and efficacy.

[0062] Example 3: In vitro activity evaluation of lysylbradykinin / bradykinin and its mutants 1. Evaluation of affinity to B2 receptor by reporter gene assay Lysylbradykinin / bradykinin exerts its pharmacological action in vivo by binding to the B2 receptor and activating the downstream signaling pathway. We constructed a cell line, B2-NFAT-CHO-K1, by stably transfecting the B2 receptor reporter gene into CHO-K1 host cells, and completed the construction of a cell library. Using this, we measured and evaluated the binding activity of lysylbradykinin / bradykinin or its mutants, and their PEG-modified derivatives to the B2 receptor in vitro.

[0063] Measurement method: 5 × 10 cells 4 The cells were plated in a 96-well plate at a density of 1000 cells / well and incubated at 37°C under 5% CO2 for 16-20 hours. Gradient diluted standard and test solutions were then added. After 5 hours, Bio-Lite Luciferase Enzyme Detection Reagent was added and the chemiluminescence intensity was measured. Graphs were created using GraphPad and a 4-parameter equation curve was fitted to obtain the EC 50 The relative activity was calculated based on the values ​​of the lysyltransferase activity. The relative affinity of the peptide mutants for the B2 receptor is shown in Table 3-1. The mutants Lys-BKm11, Lys-BKm13, Lys-BKm15 and Lys-BKm16 had a higher affinity than the wild-type lysyltransferase activity. It showed increased activity compared to radikinin.

[0064] [Table 3]

[0065] 2. Activity analysis based on major target proteins of downstream signaling pathways Using CHO-K1 cells overexpressing the B2 receptor, B2-NFAT-CHO-K1, changes in downstream signals of B2R, p-CREB and p-ERK1 / 2, were measured in vitro by WB analysis after administration of Lys-BK / BK or its different mutants. Through this, differences in the activity of the test substances were compared.C AMP-response element binding protein (CREB) is a signaling molecule that acts directly downstream after B2R activation. After stroke, activation of CREB and ERK1 / 2 can prevent post-ischemic 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.

[0066] Logarithmic growth phase CHO-K1(B2R+) cells were harvested and cultured at 3 × 10 6 Prepare a concentration of 3 x 10 cells / mL. 5 The cells were seeded in a cell culture plate at 1000 / well. The cells were cultured in a CO2 incubator at 37.0°C and 5.0% CO2 for 24-48 hours, and when the cell density reached 90% or more, the cells were washed with PBS. 1 mL 1 μM Lys-BK / BK or its mutants were added per well, and F12 medium was used as the control. The cell culture plate was incubated in a 37.0°C, 5.0% CO2 incubator for 5 minutes, and the cell culture plate was washed with PBS. Protein dissolution 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 cell debris and lysate were transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was aspirated and mixed uniformly with 5× Loading Buffer at a volume ratio of 4:1, and boiled in a metal bath at 100°C for 10 min. Equal amounts of protein samples were loaded onto 10% SDS-PAGE, followed by gel running, membrane transfer, blocking, antibody incubation, and luminescence identification.

[0067] The measurement results of the activity of Lys-BK / BK and its mutants to activate downstream signals are shown in Figure 1. Lysylbradykinin (Lys-BK) can activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. This is consistent with the mechanism of action of the drug. The in vitro biological activity of the samples was qualitatively evaluated through the determination of the phosphorylation levels of downstream ERK1 / 2 and CREB proteins, and the results showed that the mutants Lys-BKm05 and Lys-BKm07 had already lost the activity of activating the downstream pathway, while the mutants LBKm01, m02, m06, m11, m12, m13, m14, m15, and m16 all showed the activity of activating the downstream pathway.

[0068] The above two in vitro activity evaluation results show that: when lysylbradykinin / bradykinin is truncated at the C-terminus, it loses its affinity for B2R and its activity to activate downstream pathways; when lysylbradykinin is deleted from the N-terminus, it does not affect its affinity for B2R and its activity to activate downstream pathways; when other amino acids are deleted from the N-terminus, lysylbradykinin loses its affinity for B2R and its activity to activate downstream pathways; the minimum active unit is RPPGFSPFR; N-terminal extension (m11) or introduction of cysteine ​​(m13) is advantageous for increasing the affinity for B2R. As shown in Figure 1d, the phosphorylation of CREB protein in the m12, m13, and m14 groups was significantly higher than that in the LBK group, and it was revealed that m12, 13, and 14 have better in vitro activity and can activate more downstream pathways than LBK.

[0069] Example 4 Preparation of PEG conjugates of lysylbradykinin / bradykinin and its mutants The PEG modification of lysylbradykinin / bradykinin and its variants is carried out by methods known in the art. When a specific variant is modified with a specific PEG modifier, some process parameters may be slightly changed, but those skilled in the art can optimize the modification method through a limited number of experiments, and there is no technical problem. Taking the modification of wild-type BK with M-SPA-5K as an example, the preparation process is as follows:

[0070] 1. Preparation of PEG conjugate samples The peptide sample was dissolved in sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 7.0) to a final concentration of 10 mg / mL. PEG was added to the peptide solution so that the mass ratio of peptide to M-SPA-5K was 1:7, and the mixture was slowly stirred until homogenized, and then reacted at 2 to 8°C for 1 hour.

[0071] II. Purification of the reaction mixture The chromatographic conditions for the first purification step were as follows: The packing material used for purification was GE's Superdex 75 medium, and the mobile phase for purification was a 20 mM sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution with a pH of 7.0 to 7.5 (containing 0.2 M NaCl). Sample loading: After the reaction was completed, the peptide-PEG modified reaction mixture was taken and directly loaded. Elution: After sample loading, the chromatography column is washed with at least 1.5 column bed volumes of mobile phase and then eluted with UV light. 214nm Dissolution samples were collected in stages based on the trend.

[0072] The chromatographic conditions for the second stage purification were as follows: The two substances were separated by the use of a C18 reversed-phase column, which not only achieved a purification yield of over 80%, but also completely removed the free PEG.

[0073] 3. Purity analysis of PEG conjugate samples (1) Purity analysis by HPLC The measurements were performed based on the 2020 edition of the Chinese Pharmacopoeia, General Rule 0512, High Performance Liquid Chromatography. The type of chromatography used was SEC (size exclusion chromatography), the mobile phase was 20 mM PB7.0 + 10% IPA, and the chromatography column was TS. The collection conditions were 214 nm on a KG2000. RP Purity Analysis Method: The chromatography column was Protein BEH C4 2.1x150mm, 1.7μm, mobile phase A: 0.1% TFA / H2O, mobile phase B: 0.1% TFA / ACN. As shown by the results of purity analysis, the series of PEG-modified lysylbradykinin mutants prepared had a uniform product peak, no obvious impurity peaks, and a purity of >95%.

[0074] (2) Purity analysis by SDS-PAGE The purity of the samples was determined according to the SDS-polyacrylamide gel electrophoresis method of Method 5, 0541 Electrophoresis Method, in the 2020 edition of the Chinese Pharmacopoeia. Samples were measured using 12.5% ​​SDS-PAGE. As shown by the results of electrophoresis, the series of PEG-modified lysylbradykinin mutants prepared had uniform bands, no impurity bands, and a purity of >95%. In addition, since lysylbradykinin / bradykinin and its variants have a theoretically possible modification site only at the N-terminus, all of the PEG-modified products are products having a single modification at the N-terminus.

[0075] Example 5 In Vitro Evaluation of PEG Conjugates of Lysylbradykinin / Bradykinin and Its Mutants 1. Stability The stability of lysylbradykinin and its mutant PEG conjugates was indirectly evaluated in vivo by evaluating the stability in an in vitro serum system. The evaluation method was the same as in Example 2. The PEG conjugates of lysylbradykinin and its mutants all had a remaining period of 6 hours or more in an in vitro serum system (Table 5-1), and their stability was significantly improved. In the case of PEGs of the same molecular weight, the branched conjugate Lys-BKm01-YNHS10K has a higher stability than the linear conjugate Lys-BKm01-SPA10K.

[0076] [Table 4]

[0077] II. In vitro activity The affinity to B2 receptor and activity of activating downstream signal pathway of PEG conjugates of lysylbradykinin and its mutants were evaluated in vitro. The evaluation method was the same as in Example 3. Lysylbradykinin and its mutants have affinity to B2 receptor even after modification with PEG (Figures 2a and 2b), and can activate phosphorylation of key proteins in the downstream signal pathway of B2 receptor (Figure 3). Conjugates with PEG modifiers of different structural types and different molecular weights all retain their binding activity to B2 receptor well. The PEG modifiers included those with molecular weights from 2K to 20K, linear or branched, and different structures (SPA, SCM, SS, etc.). As can be seen from the evaluation results, the same Linear PEG conjugates are more active than branched PEG conjugates in terms of molecular weight. This was demonstrated for two types of PEG with molecular weights of 10K and 20K. As shown in Figure 2a, the EC 50 The activity of the linear PEG conjugate was nearly four times higher than that of the branched PEG conjugate. In addition, among the four types of PEG with the same structure, 2K, 5K, 10K, and 20K molecular weights, the 2K and 10K PEG conjugates had relatively higher activity.

[0078] As a result of evaluating the activation of downstream signaling pathways by PEG conjugates of lysylbradykinin and its variants (Figure 5), the peptides retained the activity of activating the phosphorylation of ERK1 / 2 and CREB, which are downstream signals of the B2 receptor, even after modification with PEG. Compared with the Lys-BK peptide, the above activities (especially the activity of activating ERK1 / 2 phosphorylation) were somewhat reduced. When modified with the same SPA5K-PEG modifier, Lys-BKm13-SPA5K had the highest activity among the different peptide variants. When the same peptide variant Lys-BKm01 was modified with PEG, the conjugates with molecular weights of 2K and 10K had the highest activity, and furthermore, the activity of the linear PEG conjugate Lys-BKm01-SPA10K was higher than that of the branched PEG conjugate Lys-BKm01-YNHS10K.

[0079] In conclusion, the PEG conjugates of lysylbradykinin and its variants have significantly improved stability and well retain their affinity to the B2 receptor and activity in activating downstream signaling pathways after being conjugated with PEG.

[0080] Example 6 Comparison of the efficacy of BK-PEG in the BCAS mouse model (BK is wild-type BK, PEG is SPA5K) Using a mouse model of cerebral small vessel ischemic injury caused by bilateral common carotid artery stenosis (BCAS), BK-PEG was administered intravenously several times (5 mg / kg, BK is wild-type lysylbradykinin, PEG is SPA5K), and the protective effects of repeated administration of BK-PEG against cerebral small vessel ischemic injury were observed and compared.

[0081] 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).

[0082] 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 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 chronic ischemia. The skin of the neck was sutured, and the mouse was returned to its cage and kept.

[0083] 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 (BK-PEG group).

[0084] Three days after BCAS surgery, mice were divided into groups and administered the drugs. The BCAS and SHAM groups were injected with the same volume of saline via the tail vein twice a week. The BK-PEG group (5 mg / kg) was injected with saline via the tail vein twice a week. The drug was administered by intravenous injection for a total of 7 weeks.

[0085] On the 13th and 26th postoperative days, the mice were evaluated for coordinated motor function by performing a rotarod test. The mice were placed on a mouse rotarod (diameter 30 mm × length 60 mm) in the direction opposite 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 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.

[0086] 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.

[0087] 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 testing were excluded because their data did not accurately reflect changes. Higher alternation scores indicate less impairment of working memory.

[0088] 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. A total of five tests were performed. The water temperature of the Morris water maze was maintained at 23±2°C, and the walls of the pool were marked with "East," "South," "West," and "North" to indicate the four positions for entering the water. The pool was divided into 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. To allow the mouse to identify the location of the platform, the reference object outside the water maze was not changed during the training period. 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.

[0089] On the 57th day after surgery, a sugar water preference test (SPT) was performed to evaluate the depressive behavior of the mice. Sugar water preference is an evaluation index that reflects whether or not pleasure has disappeared, while anhedonia is a loss of interest in stimuli from encouragement, and anhedonic symptoms are considered to be a manifestation of emotional disorders (including depression). The depressive state of the mice was judged based on the intake of 1% sugar water and drinking water, and the ratio of the intake of sugar water to the total amount of sugar water and drinking water was used as an index of depression in the mice, with a higher sugar water preference score indicating a lower degree of depression.

[0090] 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, Morris water maze test, and sugar water preference 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, and induced depressive behavior. In comparison between the administration group and the BCAS model group, the BK-PEG group showed statistically significant improvements in all of the above tests. This shows that the drug can significantly improve the impairment of the animals' motor coordination, forelimb muscle strength, working memory, and spatial learning and memory caused by the BCAS model, and reduce the depressive behavior caused by the BCAS model. The BCAS model is a well-accepted experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease. Therefore, the test drug can be used to treat vascular cognitive impairment, post-stroke cognitive impairment, and cerebral small vessel disease. Detailed results are summarized in Figures 4-11 and Tables 6-1-6-5.

[0091] [Table 5]

[0092] [Table 6]

[0093] [Table 7]

[0094] [Table 8]

[0095] [Table 9]

[0096] Example 7 Comparison of the efficacy of BK-PEG in the BCAS mouse model (BK is m11, PEG is SPA5K) Using a mouse model of cerebral small vessel ischemic injury caused by bilateral common carotid artery stenosis (BCAS), BK-PEG was administered intravenously several times (5 mg / kg, BK was m11, PEG was SPA5K), and the protective effects of repeated administration of BK-PEG against cerebral small vessel ischemic injury were observed and compared.

[0097] I. Group assignment and experimental design A bilateral common carotid artery spring constriction procedure was performed to prepare a mouse model of cerebral small vessel ischemia injury caused by bilateral common carotid artery stenosis (BCAS). The method for preparing the BCAS cerebral small vessel injury model was the same as in Example 6.

[0098] A total of two groups were set up: a model group (BCAS group) and a BK-PEG (5 mg / kg) group. Seven days after BCAS surgery, the animals were divided into groups and administered the drug. The model group (BCAS group) was injected with the same volume of saline via the tail vein twice a week. The BK-PEG group was injected with the drug via the tail vein twice a week for a total of 6 weeks. Six weeks after administration, the mice were evaluated for their coordinated motor function by performing a Rotarod test. One week after the end of administration, the mice were evaluated for their spatial learning and memory impairment by performing a Morris water maze test.

[0099] 2. Results Compared with the model group (BCAS group), the BK-PEG group showed a tendency of improvement in the results of the rotarod test, and a statistically significant difference was observed in the results of the Morris water maze test. This indicated that the drug could improve the motor coordination and spatial learning and memory impairment of animals induced by the BCAS model. The BCAS model is a well-accepted experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease. Therefore, the test drug is considered to be usable for the treatment of vascular cognitive impairment, post-stroke cognitive impairment, and cerebral small vessel disease. The detailed results are summarized in Tables 10-11.

[0100] [Table 10]

[0101] [Table 11]

[0102] Example 8 Comparison of the efficacy of BK-PEG in the BCAS mouse model (BK is m13, PEG is SPA2K) Using a mouse model of cerebral small vessel ischemic injury caused by bilateral common carotid artery stenosis (BCAS), BK-PEG was administered subcutaneously several times (15 mg / kg, BK was m13, PEG was SPA2K), and the protective effects of repeated administration of BK-PEG against cerebral small vessel ischemic injury were observed and compared.

[0103] I. Group assignment and experimental design A bilateral common carotid artery spring constriction procedure was performed to prepare a mouse model of cerebral small vessel ischemia injury caused by bilateral common carotid artery stenosis (BCAS). The method for preparing the BCAS cerebral small vessel injury model was the same as in Example 6.

[0104] A total of two groups were set up: a model group (BCAS group) and a BK-PEG (15 mg / kg) group. Seven days after BCAS surgery, subjects were divided into groups and administered the drug. The model group (BCAS group) received the same volume of saline subcutaneous injection twice a week. The BK-PEG group received the drug subcutaneously twice a week for a total of 8 weeks. Eight weeks after administration, the mice were evaluated for their coordinated motor function by performing the Rotarod test. One week after the end of administration, the mice were evaluated for their spatial learning and memory impairment by performing the Morris water maze test.

[0105] 2. Results Compared with the model group (BCAS group), the BK-PEG group showed a tendency of improvement in the results of the rotarod test, and a statistically significant difference was observed in the results of the Morris water maze test. This indicated that the drug could improve the motor coordination and spatial learning and memory impairment of animals induced by the BCAS model. The BCAS model is a well-accepted experimental animal model in the study of vascular cognitive impairment and cerebral small vessel disease. Therefore, the test drug is considered to be usable for the treatment of vascular cognitive impairment, post-stroke cognitive impairment, and cerebral small vessel disease. The detailed results are summarized in Tables 12-13.

[0106] [Table 12]

[0107] [Table 13]

[0108] In Examples 6 to 8, 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. The test drug used in the Examples was bradykinin, which retains affinity to bradykinin receptors and activity of downstream signal pathway activation, and after administration to BCAS model mice, the animals' impaired coordinated motor function, impaired muscle strength in the forelimbs, working memory, spatial learning and memory function, and depressive behavior were improved.

[0109] In addition to the test drugs of Examples 6 to 8, similar effects were observed when other bradykinin derivatives (e.g., Lys-BKm01, m02, m06, m12, m14, and other suitable PEG conjugates) that retained the affinity to the bradykinin receptor and the activity of activating downstream signal pathways were administered to BCAS model mice. That is, improvements were observed in the impaired coordinated motor function, impaired muscle strength of the forelimbs, working memory, spatial learning and memory function, and depressive behavior in BCAS model mice. Conversely, no similar effects were observed when bradykinin derivatives (e.g., PEG conjugates such as Lys-BKm05 and m07) that had relatively lost the affinity to the bradykinin receptor and the activity of activating downstream signal pathways were administered.

[0110] In summary, any bradykinin derivative that retains good receptor affinity and downstream signal pathway activation activity can be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

[0111] In addition, peptide drugs generally have problems of poor stability in vivo and short half-life. Lysylbradykinin and bradykinin are more unstable than normal peptides, with half-lives of only a few seconds, and exogenously administered kinins are quickly hydrolyzed and inactivated by kininase in vivo, so they have no druggability. Therefore, only long-acting lysylbradykinin and bradykinin have druggability. Although the long-acting methods exemplified in the examples are all conjugated with PEG, making lysylbradykinin and bradykinin long-acting is not the main problem to be solved in the present invention, and the PEG conjugation technology used in the specific examples is for illustrative purposes and does not limit the long-acting method of the present invention. Those skilled in the art will appreciate that lysylbradykinin / bradykinin derivatives that retain the affinity to the receptor and the activity of activating downstream signal pathways are available in other forms. Even if the method is used for a long time, it is expected that it will improve the damaged functions of BCAS model mice and be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

[0112] Furthermore, the examples illustrate the stability, in vitro activity, and in vivo efficacy of PEG-modified lysylbradykinin or bradykinin and various variants, all of which show good stability, and all of the lysylbradykinin or bradykinin variants that retain their in vitro activity can be extended in half-life after being modified with PEG, and can simultaneously exert their in vivo biological activity. Meanwhile, other lysylbradykinin or bradykinin variants that do not retain their in vitro activity, after being modified with PEG, or these lysylbradykinin or bradykinin variants themselves (not modified with PEG), all showed no in vivo efficacy. Therefore, those skilled in the art can reasonably predict that, without being limited to the specific lysylbradykinin or bradykinin mutants exemplified in the examples, other lysylbradykinin or bradykinin derivatives that retain receptor binding activity will all have an extended half-life of lysylbradykinin or bradykinin after being made to act for a long time, and at the same time be able to exert biological activity in vivo, and can improve the damaged functions of BCAS model mice and be used to treat vascular cognitive dysfunction, post-stroke cognitive dysfunction, and cerebral small vessel disease.

Claims

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

2. A pharmaceutical composition for treating or ameliorating vascular cognitive impairment, comprising kinin or a derivative thereof.

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

4. Use of quinine or a derivative thereof in the manufacture of a medicament for treating or ameliorating post-stroke cognitive impairment.

5. A pharmaceutical composition for treating or ameliorating post-stroke cognitive impairment, comprising kinin or a derivative thereof.

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

7. Use of quinine 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 kinin or a derivative thereof.

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

10. Use of quinine or a derivative thereof in the manufacture of a medicament for treating or ameliorating cerebral small vessel disease.

11. A pharmaceutical composition for treating or ameliorating cerebral small vessel disease, comprising kinin or a derivative thereof.

12. A method for treating or ameliorating cerebral small vessel disease, comprising administering to a patient a kinin or a derivative thereof.

13. The kinin according to any one of claims 1 to 12, characterized in that it is bradykinin or lysylbradykinin.

14. The quinine derivative according to any one of claims 1 to 12, characterized in that it is a long-acting quinine.

15. 15. A quinine derivative according to claim 14, the quinine derivative is a polyethylene glycol modified quinine, The PEG modifier used is an SPA, SCM, or SS modifier, and is a linear or branched PEG modifier; Quinine derivatives.

16. 15. A quinine derivative according to claim 14, The quinine derivative is a mutant of quinine, Introducing cysteine ​​at any position in the amino acid sequence of lysylbradykinin or bradykinin, and / or inserting any amino acid between the 1st and 2nd amino acid residues of the N-terminus of lysylbradykinin; and / or inserting any amino acid before the first amino acid residue at the N-terminus of bradykinin; and / or two or more lysylbradykinin molecules, or two or more bradykinin molecules, or a lysylbradykinin molecule and a bradykinin molecule linked together; and / or characterized in that Phe at position 6 of lysylbradykinin or Phe at position 5 of bradykinin is mutated to another amino acid; Quinine derivatives.

17. 15. A quinine derivative according to claim 14, The kinin derivative is a fusion protein, characterized in that the kinin derivative is fused to a kinin and an Fc fragment or fused to a kinin and human serum albumin. Quinine derivatives.

18. The quinine derivative according to claim 14, characterized in that the quinine derivative is a fatty acid-added quinine or a long-acting formulation of quinine.

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