Polyethylene glycol modified quinine or its derivatives and their use in medicine

Abstract

A polyethylene glycol modified kinine or its derivative and its use in drugs. The present invention uses PEG modification technology to significantly extend the half-life of kinine and simultaneously exert its biological activity, solving the problem of lack of druggability due to a short half-life. PEG modifiers include, but are not limited to, SPA, SCM, and SS modifiers. They may be linear or branched PEG modifiers. The modified kinine mutant of the present invention has an extended half-life while retaining its affinity to the receptor, and further has a better efficacy after PEG modification than wild-type kinine, further improving the druggability of kinine. The PEG modified kinine of the present invention exhibits remarkable drug efficacy even when administered by various routes. In particular, subcutaneous injection and oral administration are convenient for clinical use, effectively improve patient compliance, and are suitable for long-term treatment administration such as the recovery treatment of stroke patients.

Description

[Technical field]

[0001] The present invention relates to polyethylene glycol-modified quinine or a derivative thereof and its use in medicine, and in particular to improving the druggability of quinine by PEG modification technology. [Background technology]

[0002] The kallikrein-kinin system (KKS) is an important regulatory system in the body and is involved in various physiological and pathological processes, such as regulating the functions of the cardiovascular system, kidney, and nervous system. The KKS system includes kallikrein (KLK), kininogen, kinin, kinin receptors (B1 and B2 receptors), and kininase. Among them, kallikrein and kinin are the core components. 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 effects. Kinin is a type of active peptide that exists in very small amounts in the human body. Kinins that have been well studied include lysylbradykinin (Lys-BK), bradykinin (BK), and angiotensin.

[0003] Lysylbradykinin is mainly produced by hydrolysis of low molecular weight kininogen LMWK catalyzed by KLK1, and consists of 10 amino acids with the sequence KRPPGFSPFR. Lysylbradykinin is cleaved by aminopeptidases in vivo to form bradykinin (9 peptide units of RPPGFSPFR), and both have a wide range of physiological functions, including regulation of blood pressure and inflammation. Specifically, they bind to G protein-coupled receptors, activate the phosphorylation of extracellular signal-regulated protein kinases 1 / 2 (ERK1 / 2) and cAMP-response element binding protein (CREB), stimulate the release of second messengers such as nitric oxide (NO), cAMP, and prostacyclin I2, and exert biological effects such as dilation of small arteries, increased local blood flow, increased vascular permeability, and vasodilation through antagonism of the renin-angiotensin system (RAS).

[0004] The degradation of lysylbradykinin in vivo is mainly involved in aminopeptidase, carboxypeptidase and angiotensin ACEII, and the dynamic balance of bradykinin in vivo is maintained by the kinin releasing enzyme system and the decomposing enzyme system. The exogenous administration of lysylbradykinin and bradykinin relaxes blood vessels and has the potential to be applied to the treatment of cerebrovascular diseases. However, the half-life of lysylbradykinin and bradykinin in vivo is extremely short, only a few seconds, and they are quickly inactivated by hydrolysis of kinases, so their short biological half-life greatly limits their druggability.

[0005] At present, the main strategies for making peptide drugs long-acting include chemical modification (including polyethylene glycol modification, peptide main chain terminal modification, side chain modification, cyclization, amino acid substitution, glycosylation modification, etc.), microsphere embedding (micro- and nanoparticle embedding, including lipid carrier nanoparticles, polymer carrier nanoparticles, and inorganic carrier nanoparticles), and protein fusion technology (currently, the most commonly used fusion protein is albumin and immunoglobulin (IgG)). At present, various methods are used, including: Although each method has its own advantages and disadvantages, it has been reported that the application potential of the micro- and nanoparticle embedding method is relatively high (Cheng Nian, Hu Zhongping et al. Research progress on long-acting peptide drugs [J]. China New Drug Journal, 2016, Vol. 25, No. 22).

[0006] Removing enzyme cleavage sites by amino acid substitution to prevent peptides from being degraded is an effective way to extend the half-life of peptides in vivo. Lobradimil is a drug in which the 3rd (Pro), 5th (Phe), 8th (Phe), and 9th (Arg) positions of the amino acid sequence of BK are mutated to unnatural amino acids, respectively, to extend the half-life of BK in vivo. Its main effect is to increase the permeability of the blood-brain barrier and treat pediatric brain tumors in combination with carboplatin-based chemotherapy. Although the safety was relatively good, the efficacy did not reach the endpoint (Cancer Chemother. Pharmacol. 2006,58:343-347), and the project was discontinued after the completion of phase 2 clinical trials. Icatibant, which was approved for sale by the FDA in 2011, is also a drug based on bradykinin. This is a drug whose in vivo half-life has been extended by mutating the 3rd (Pro), 5th (Phe), 7th (Pro), and 8th (Phe) amino acid sequence of BK to unnatural amino acids, respectively, and its indication is a rare disease called hereditary angioedema. This drug molecule is a selective competitive antagonist of the B2 receptor and has the opposite effect to bradykinin. Currently, there are no reports of successful clinical application of the vasodilatory effect of extending the in vivo half-life of LBK / BK by amino acid mutation.

[0007] Modification with polyethylene glycol (PEG) is one of the methods to extend the half-life and improve the stability of proteins in vivo. There are currently more than a dozen PEG-modified protein drugs on the market. However, there are still few PEG-modified peptide drugs. Peptides generally exert their biological functions by binding to receptors in the body. Because peptide molecules are small, PEG modification may affect the binding of peptides to receptors. As a result, the biological activity of peptides is lost. For example, bradykinin consists of only nine amino acids and has a small molecular structure. There have been no research reports on polyethylene glycol-modified lysylbradykinin or bradykinin at home or abroad. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Cheng Nian, Hu Zhongping et al. Research progress on long-acting polypeptide drugs [J]. China New Drug Journal, 2016, Vol. 25, No. 22 [Non-Patent Document 2] Cancer Chemother. Pharmacol. 2006,58:343-347 Summary of the Invention [Problem to be solved by the invention]

[0009] The technical problem to be solved by the present invention is to extend the half-life of lysylbradykinin or bradykinin, while at the same time exerting its biological activity in vivo, and resolving the problem of its druggability. [Means for solving the problem]

[0010] On the one hand, the present invention uses PEG modification technology to significantly extend the half-life of lysylbradykinin or bradykinin, while at the same time allowing it to exert its biological activity in vivo.

[0011] The polyethylene glycol-modified lysylbradykinin or bradykinin provided by the present invention is modified by N-terminal PEG, not by C-terminal, and the lysylbradykinin or bradykinin is a wild-type lysylbradykinin or bradykinin, or is modified (non-polyethylene glycol-modified) based on the wild-type lysylbradykinin or bradykinin. The derivatives include not only the various mutants exemplified in the examples of the present invention, but also peptides obtained by further modifying 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 modifications (excluding polyethylene glycol modification). The derivatives retain the in vitro activity (receptor binding activity) of wild-type lysylbradykinin or bradykinin. In the examples, the stability, in vitro activity, and in vitro activity of PEG-modified lysylbradykinin or bradykinin and several mutants thereof were examined. As a result of verifying the efficacy in vivo, all of them showed good stability. In addition, after the lysylbradykinin or bradykinin mutants that retain their in vitro activity are modified with PEG, the half-life of the lysylbradykinin or bradykinin can be extended and the biological activity in vivo can be exhibited at the same time. On the other hand, after the other lysylbradykinin or bradykinin mutants that do not retain their in vitro activity are modified with PEG, or the lysylbradykinin or bradykinin mutants themselves (not modified with PEG), none of them showed any efficacy in vivo. Therefore, those skilled in the art can reasonably expect that, without being limited to the specific lysylbradykinin or bradykinin mutants exemplified in the examples, other lysylbradykinin or bradykinin derivatives that retain their receptor binding activity can be modified with polyethylene glycol and the half-life of the lysylbradykinin or bradykinin can be extended and the biological activity in vivo can be exhibited at the same time.

[0012] PEG modifiers include, but are not limited to, SPA, SCM, and SS modifiers. PEG modifiers may be linear or branched. Preferably, the PEG modifier is a linear PEG modifier. Preferably, the molecular weight of the PEG modifier is from 2 KD to 20 KD. Preferably, the molecular weight of the PEG modifier is from 2 KD to 10 KD. Preferably, the molecular weight of the PEG modifier is 2KD or 10KD.

[0013] On the other hand, the present invention further improves the druggability of lysylbradykinin or bradykinin by optimizing the amino acid sequence thereof.

[0014] The present invention provides a derivative of lysylbradykinin or bradykinin obtained by introducing cysteine ​​(Cys) into any position in the amino acid sequence of wild-type lysylbradykinin or bradykinin. Lysylbradykinin or bradykinin into which cysteine ​​has been introduced has shown significant advantages in in vivo pharmacological evaluation. 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.

[0015] The present invention also provides a lysylbradykinin derivative obtained by inserting any number and any type of amino acid between the 1st and 2nd amino acid residues of the N-terminus of wild-type lysylbradykinin, i.e., KXRPPGFSPFR, and a bradykinin derivative obtained by inserting any number and any type of amino acid before the 1st amino acid residue of the N-terminus of wild-type bradykinin, i.e., XRPPGFSPFR. X represents any number and any type of amino acid. Experimental results have shown that lysylbradykinin or bradykinin with an extended N-terminus has better activity. Preferably, 1, 2, or 3 amino acids are inserted. Preferably, 1 or 2 arginines (Arg) are inserted.

[0016] The present invention also provides a lysylbradykinin or bradykinin derivative (e.g., m14, m12) in which two or more lysylbradykinin units are linked together, or two or more bradykinin units are linked together, or a lysylbradykinin unit and a bradykinin unit are linked together. The lysylbradykinin or bradykinin unit may be a wild-type lysylbradykinin or may be bradykinin, or may be a mutant obtained by substituting, inserting or deleting a portion of amino acids on the basis of wild-type lysylbradykinin or bradykinin.

[0017] The present invention also provides a lysylbradykinin or bradykinin derivative in which Phe at position 6 of wild-type lysylbradykinin or Phe at position 5 of wild-type bradykinin is mutated to another amino acid, such as the unnatural amino acid Igl (α-2-indolylglycine) or 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.

[0018] Preferably, the amino acid sequence of the lysylbradykinin or bradykinin derivative of the present invention is the sequence shown in SEQ ID NO:14.

[0019] The present invention also provides compositions comprising said lysylbradykinin or bradykinin derivatives.

[0020] Lysylbradykinin or bradykinin acts on the corresponding receptor to exert a series of physiological effects. However, lysylbradykinin and bradykinin have a very short half-life of several seconds in vivo, and are quickly inactivated by hydrolysis with kininase, so their druggability is greatly limited by their short biological half-life. The present invention uses polyethylene glycol modification technology to greatly extend the half-life of lysylbradykinin or bradykinin in vivo, and at the same time, it exerts the in vitro and in vivo biological activity of lysylbradykinin or bradykinin well, and exhibits the in vivo activity of improving cerebral infarction symptoms in MCAO cerebral ischemia reperfusion model, and solves the problem that lysylbradykinin or bradykinin alone has too short a half-life to be druggable.

[0021] The present invention provides the use of polyethylene glycol-modified lysylbradykinin or bradykinin in the manufacture of a drug for the treatment, prevention, prognosis improvement and recurrence prevention of ischemic stroke. The lysylbradykinin or bradykinin may be a wild-type lysylbradykinin or bradykinin, or a derivative obtained by modification (non-polyethylene glycol modification) based on the wild-type lysylbradykinin or bradykinin. The derivative retains the in vitro activity (i.e., binding activity to a receptor) of the wild-type lysylbradykinin or bradykinin, and includes not only various mutants exemplified in the examples of the present invention, but also peptides obtained by further modification based on the Lys-BK / BK mutant of the present invention, fusion proteins (including but not limited to albumin fusion, Fc fusion, etc.), and various types of modifications (excluding polyethylene glycol modifications).

[0022] Preferably, the lysylbradykinin or bradykinin variants and their PEG conjugates are administered orally by injection.

[0023] Compared with the prior art, the present invention has the following advantages: First, the present invention uses polyethylene glycol modification technology to greatly extend the half-life of lysylbradykinin or bradykinin in vivo, while at the same time making lysylbradykinin or bradykinin's biological activity well manifest, solving the problem that lysylbradykinin or bradykinin alone has a too short half-life and is not druggable. Regarding in vivo efficacy, PEG-modified lysylbradykinin or bradykinin and its variants showed a significant improvement effect on neurological deficit symptoms in MCAO cerebral ischemia-reperfusion model, and a significant improvement effect on cerebral infarction area.

[0024] Second, the modified lysylbradykinin or bradykinin mutants of the present invention have a distinct advantage: they are stable in serum systems while retaining affinity for the receptor. The in vivo efficacy of these mutants was significantly superior to that of wild-type lysylbradykinin, suggesting a longer half-life in vivo, and after modification with PEG, they showed superior in vivo efficacy to wild-type lysylbradykinin or PEG-modified bradykinin.

[0025] Thirdly, the PEG-modified lysylbradykinin or bradykinin of the present invention can exert a remarkable medicinal effect even when administered by various administration routes such as intravenous injection, subcutaneous injection, oral administration, etc. In particular, subcutaneous injection and oral administration are convenient for clinical use because patients do not need to go to the hospital regularly to receive injections and can self-administer at home, effectively improving patient compliance, and are more suitable for long-term therapeutic administration such as the treatment of stroke patients in the recovery period. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the UPLC-RP purity analysis profile of the peptide variant Lys-BKm13. [Figure 2a]Figure 2 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 2b] Figure 2 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 2c] Figure 2 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 2d] Figure 2 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. [Diagram 3] FIG. 3 shows the UPLC-RP purity analysis profile of the Lys-BKm13-SPA10K sample. [Figure 4a] FIG. 4 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 4b]FIG. 4 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 5] Figure 5 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 6] FIG. 6 shows the drug concentration-time curve of Lys-BKm13-SPA10K in the body of an SD rat. [Figure 7a] FIG. 7 is a graph showing the effects of the test substance of Example 6 on neurological deficit symptoms and cerebral infarction area. [Figure 7b] FIG. 7 is a graph showing the effects of the test substance of Example 6 on neurological deficit symptoms and cerebral infarction area. [Figure 8a] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8b] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8c] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8d] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8e] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8f] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8g] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8h] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8i]FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8j] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8k] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 8l] FIG. 8 is a graph showing the effects of the test substance of Example 7 on neurological deficit symptoms and cerebral infarction area. [Figure 9] FIG. 9 is a graph showing the effects of the test substance of Example 8 on neurological deficit symptoms and cerebral infarction area. [Figure 10a] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 10b] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 10c] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 10d] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 10e] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 10f] FIG. 10 shows photographs of brain sections from the animals in Example 8. [Figure 11a] FIG. 11 is a graph showing the effects of the test substance of Example 9 on neurological deficit symptoms and cerebral infarction area. [Figure 11b] FIG. 11 is a graph showing the effects of the test substance of Example 9 on neurological deficit symptoms and cerebral infarction area. [Figure 12a] FIG. 12 shows photographs of brain sections from the animals in Example 9. [Figure 12b] FIG. 12 shows photographs of brain sections from the animals in Example 9. [Figure 12c] FIG. 12 shows photographs of brain sections from the animals in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] 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.

[0028] BK: wild-type bradykinin, which is identical to the amino acid sequence of native human bradykinin, and whose amino acid sequence is RPPGFSPFR.

[0029] Mutant: A mutant obtained by substituting, inserting or deleting some amino acids on the basis of wild-type lysylbradykinin or bradykinin.

[0030] 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 modifications (excluding polyethylene glycol modification).

[0031] 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).

[0032] 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.

[0033] 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.

[0034] [ka]

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] [ka]

[0040] 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.

[0041] [ka]

[0042] 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.

[0043] [ka] EXAMPLES

[0044] Example 1 Design and synthesis of mutants of lysylbradykinin / bradykinin A series of mutants were designed based on the sequence of lysylbradykinin / bradykinin and possible degradation pathways in vivo (Table 1-1). The mutants were mainly those in which amino acids in the sequence of lysylbradykinin / bradykinin were replaced with structurally similar natural or non-natural amino acids (m01-m04, m07, and m02 are BK mutants), and those that were not lysylbradykinin / bradykinin. These include partially truncated or extended peptide mutants (m05, m06, m08-m11) designed to identify the minimum active unit that exerts the biological activity / function of radikinin, mutants (m13, m15, m16) into which cysteine ​​has been introduced to further enhance the antioxidant power 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 pharmacological efficacy.

[0045] [Table 1]

[0046] The measurement was performed with reference to the 2020 edition of the Chinese Pharmacopoeia, General Rule 0512 High Performance Liquid Chromatography Method. The type of measurement chromatography was C18-RP (reverse phase chromatography), the mobile phase was A: 0.1% TFA / H2O; B: 0.1% TFA / ACN, the chromatography column was ACQUITY BEH C18: (1.7 μm, 2.1 × 50 mm), and the collection conditions were 214 nm. As shown by the measurement results, the purity of each peptide was all > 95%. Taking Lys-BKm13 as an example, the purity analysis chromatogram is shown in Figure 1.

[0047] 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 degradation of peptide samples in serum systems was monitored based on the method. The stability of the tide samples was evaluated. 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.

[0048] [Table 2]

[0049] 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.

[0050] 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.

[0051] Measurement method: 5 × 10 cells 4 Plated in a 96-well plate at a density of 37 The cells were incubated at ℃ and 5% CO2 for 16-20 hours. Then, the standard and test solutions diluted in a concentration gradient were added. After 5 hours, Bio-Lite Luciferase Enzyme Detection Reagent was added and the chemiluminescence intensity was measured. Graphs were created in GraphPad and a four-parameter equation curve was fitted to obtain the EC 50 The relative activity was calculated based on the values. The relative affinity of the peptide variants for the B2 receptor is shown in Table 3-1, and variants Lys-BKm11, Lys-BKm13, Lys-BKm15 and Lys-BKm16 showed increased activity compared to wild-type lysylbradykinin.

[0052] [Table 3]

[0053] 2. Activity analysis based on major target proteins of downstream signaling pathways Based on the B2R-overexpressing CHO-K1 cells, B2-NFAT-CHO-K1, The changes in downstream signals of B2R, p-CREB and p-ERK1 / 2, due to administration of Lys-BK / BK or its different mutants in vitro were measured by WB method. Through this, the 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.

[0054] 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 in a volume ratio of 4:1, and boiled in a metal bath at 100°C for 10 minutes. The pool was loaded onto 10% SDS-PAGE and subjected to operations such as gel running, membrane transfer, blocking, antibody incubation, and luminescence identification.

[0055] The measurement results of the activity of Lys-BK / BK and its mutants to activate downstream signals are shown in Figure 2. Lysylbradykinin (Lys-BK) can activate the phosphorylation of downstream ERK1 / 2 and CREB proteins, which 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. 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.

[0056] 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.

[0057] 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:

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 3. Purity analysis of PEG conjugate samples (1) Purity analysis by HPLC The measurement was performed with reference to the 2020 edition of the Chinese Pharmacopoeia, General Rule 0512, high performance liquid chromatography method. The type of measurement chromatography was SEC (size exclusion chromatography), the mobile phase was 20 mM PB7.0 + 10% IPA, the chromatography column was TSK G2000, and the collection conditions were 214 nm. 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 the purity analysis, the PEG-modified lysylbradykinin mutants we prepared had a uniform product peak, no obvious impurity peaks, and a purity of >95%. Taking Lys-BKm13-SPA10K as an example, its purity analysis chromatogram is shown in Figure 3.

[0062] (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.

[0063] 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.

[0064] [Table 4]

[0065] II. In vitro activity The affinity and affinity for B2 receptors of lysylbradykinin and its mutants conjugates with PEG were examined. The activity of lysylbradykinin and its mutants to bind to B2 receptor and activate downstream signal pathways was evaluated in vitro. The evaluation method was the same as in Example 3. Lysylbradykinin and its mutants still have affinity for B2 receptor after modification with PEG (Fig. 4a, 4b), and can activate phosphorylation of key proteins in downstream signal pathways of B2 receptor (Fig. 5). Conjugates with PEG modifiers of different structure types and different molecular weights all retain their binding activity to B2 receptor. The PEG modifiers included PEG modifiers 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, linear PEG conjugates have higher activity than branched PEG conjugates at the same molecular weight. This is shown for two types of PEG with molecular weights of 10K and 20K. As shown in Figure 4a, the EC of Lys-BKm01-SPA10K and Lys-BKm01-YNHS10K was 100%. 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.

[0066] The activation of downstream signaling pathways by PEG conjugates of lysylbradykinin and its mutants was evaluated (Figure 5). After modification with PEG, the peptides retained the activity of activating the phosphorylation of ERK1 / 2 and CREB, downstream signals of the B2 receptor. 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 mutants. When the same peptide mutant Lys-BKm01 was modified with PEG, the activity of the conjugates with PEGs of molecular weights 2K and 10K was the highest, and the activity of the linear PEG conjugate Lys-BKm01-SPA10K was higher than that of the branched PEG conjugate Lys-BKm01-YNHS10K.

[0067] Example 6 In vivo metabolism study of lysylbradykinin-PEG conjugates 1. Experimental Method SD rats were subcutaneously administered 0.5 mg / kg of lysylbradykinin-PEG conjugate (Lys-BKm13-SPA10K as an example), and plasma was collected before administration and 5, 15, 0.5, 1, 2, 4, 8, 24, 48, and 72 hours after administration. The content of Lys-BKm13-SPA10K in the plasma samples was measured by specific ELISA (unit: ng / ml).

[0068] 2.Measurement method 1. Coating: Coat the microplate with streptavidin (4ug / ml) and leave it at 2-8℃ overnight. Wash once with washing solution, add blocking solution, block at 37℃ for 2 hours, and set aside for later use. 2. Capture material: Commercially available anti-PEG-biotin was diluted to 0.5ug / ml with blocking solution and added to the above microplate. After 2 hours at 37℃ and 300rpm, it was washed three times with washing solution and tapped to dry. 3. Sample loading: The standard or sample was diluted to the corresponding concentration, and the diluted standard or sample was diluted 10 times with blocking solution and added to the above microplate. After 1.5 hours at 37℃ and 300 rpm, it was washed three times with washing solution and tapped dry. 4. Detection material: HRP-labeled anti-Lys-BKm13-SPA10K specific antibody was diluted 1000 times and added to the above microplate. After 1.5 hours at 37℃ and 300 rpm, it was washed three times with washing solution and tapped dry. 5. Color development: TMB was added to the above microplate, and the color was developed at 37°C for 15 minutes, after which 2M H2SO4 was added. 6. Plate reading: OD measured using a microplate reader 450 and O.D. 630 It was read as follows. Concentrations of drugs in samples were calculated with a four-parameter fit using OriginPro 9.1.

[0069] 3. Measurement results The drug concentration-time curve of Lys-BKm13-SPA10K in SD rats is shown in Figure 6. SD rats were subcutaneously administered once (0.5 mg / kg), and both animals (one male and one female) showed C max C max were 88.07ng / ml and 114.68ng / ml, respectively. A study has shown that after 30mg of icatibant (a structural analogue of bradykinin) was administered subcutaneously to adults, the blood drug concentration peaked (405ng / ml) in 1.79 hours (reference: Efficacy, pharmacokinetics, and safety of icatibant for the treatment of Japanese patients with an acute attack of hereditary angioedema: A phase 3 open-label study). When comparing equivalent doses based on body surface area, Lys-BKm13-SPA10K had a higher C maxand the time to peak was longer, suggesting that Lys-BKm13-SPA10K has a higher bioavailability.

[0070] Example 7 In vivo activity evaluation of lysylbradykinin (non-mutated)-PEG conjugates 1. Creating a model SD rats, male, SPF grade, weighing 270-300g, were used. A middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model was prepared using the middle cerebral artery embolization method. The animals were anesthetized with isoflurane, fixed in a supine position, the skin was disinfected, the midline of the neck was incised, the right common carotid artery, external carotid artery, and internal carotid artery were isolated, the vagus nerve was gently dissected, and the external carotid artery was ligated and cut. The proximal end of the common carotid artery was clamped, an incision was made distal to the ligated part of the external carotid artery, and an MCAO embolization plug (model number 2438-A5, purchased from Beijing Seino Technology Co., Ltd.) was inserted, passed through the common carotid artery bifurcation and entered the internal carotid artery, and gradually inserted until a slight resistance was felt (approximately 20 mm from the bifurcation), at which point the plug stopped and the blood flow of the middle cerebral artery was blocked. The neck skin was sutured and disinfected, and the rat was returned to its cage. After 90 minutes of ischemia, the rat was anesthetized again, fixed to a board, the neck skin was incised, the thrombus was located and gently removed, and blood flow was restored to allow reperfusion. The neck skin was sutured and disinfected, and the rat was returned to its cage for housing.

[0071] Grouping and administration: Sham-operated group, model group (administered PBS by intravenous injection), edaravone group (administered by intravenous injection at 6 mg / kg), lysylbradykinin-PEG complex group (administered by intravenous injection at 3 mg / kg, dose calculated based on peptide) were set up. The administration group was administered once by injection 2 hours after reperfusion.

[0072] 2. Evaluation of neurological deficit symptoms [Table 5]

[0073] 3. Measurement of cerebral infarction volume The animals were anesthetized with 10% chloral hydrate, the heads were cut, the brains were extracted, the olfactory bulbs, cerebellum and low brain stem were removed, blood stains on the cerebral surface were washed away with saline, the residual water on the surface was blotted, and the brains were left at -80°C for 7 minutes. After that, immediately after removal, coronal sections were cut downward perpendicular to the cross plane of the line of sight, and further sections were cut posteriorly every 2 mm. The brain slices were placed in a TTC (20 g / L) staining solution freshly prepared with saline and warmed at 37°C for 90 minutes, whereby normal brain tissue was stained deep red and ischemic brain tissue was pale. After rinsing with saline, the brain slices were immediately arranged in order from front to back, the residual water on the surface was blotted, and photographed. Statistics were taken based on the photographs using image analysis software (Image Tool), and the right ischemic area (white area) and the right area were surrounded to calculate the percentage of cerebral infarction area using the following formula: Calculation formula: cerebral infarction area% = total ischemic area ÷ total right area × 100.

[0074] 4. Results 1. Effect of the test substance on neurological deficit symptoms As shown in Figure 7a, the positive drug group (Edaravone) had a significant improvement effect on nerve deficiency symptoms compared to the model group. In addition, the lysylbradykinin-PEG conjugate Lys-BK-SPA5 group had a significant improvement effect on nerve deficiency symptoms compared to the model group, and the difference was statistically significant.

[0075] [Table 6]

[0076] 2. Effect of the test substance on cerebral infarction area (%) As shown in Figure 7b, the positive drug group (Edaravone) had a significant improvement effect on the cerebral infarction area compared to the model group. Also, the lysylbradykinin-PEG conjugate group had a significant improvement effect on the cerebral infarction area compared to the model group, and the difference was statistically significant.

[0077] [Table 7]

[0078] Example 8. Evaluation of in vivo activity of lysylbradykinin / bradykinin mutant-PEG conjugates Lysylbradykinin and its variants PEG conjugates were evaluated for efficacy in vivo in the MCAO model several times. All experiments except Table 7-6 were administered at a dose of 3 mg / kg (all doses in the administration groups were calculated based on the peptide), and the administration method was intravenous or subcutaneous injection, with a single injection administered 2 hours after reperfusion. Other specific experimental protocols were performed with reference to Example 7. The experimental results are shown in Figure 8 and Table 7.

[0079] [Table 8]

[0080] [Table 9]

[0081] [Table 10]

[0082] [Table 11]

[0083] [Table 12]

[0084] [Table 13]

[0085] Example 9 In vivo administration of conjugates with different PEG modifiers via different routes of administration Activity evaluation in In this example, the efficacy of the lysylbradykinin mutant-PEG conjugate was examined in the same model when administered by intravenous or subcutaneous injection. The dose was 4.5 mg / kg (calculated based on the peptide), and other specific experimental protocols were performed with reference to Example 7. The experimental results are shown in Figures 9, 10, and Table 8-1. When administered by intravenous or subcutaneous injection, the Lys-BKm13-SPA5K group had a significant improvement effect on both neurological deficit symptoms and cerebral infarction area compared to the model group, and the difference was statistically significant. As a result of evaluating the efficacy of conjugates using PEG modifiers with different molecular weights, Lys-BKm01-SPA2K has a significantly superior in vivo efficacy to Lys-BKm01-SPA5K.

[0086] [Table 14]

[0087] Example 10: Evaluation of in vivo activity of oral administration of lysylbradykinin / bradykinin mutant-PEG conjugates In the MCAO model, PEG conjugates of lysylbradykinin and PEG conjugates of lysylbradykinin mutants were orally administered to compare their in vivo efficacy. In this example, lysylbradykinin is unmutated wild-type lysylbradykinin, the mutant is M01, and PEG is M-SPA-5K. Administration was performed at a single dose of 6 mg / kg (calculated based on the peptide). The results of the experiment are shown in Figure 11, Figure 12, and Table 9-1.

[0088] 1. Effect of the test substance on neurological deficit symptoms As shown in Table 9-1, the PEG conjugate of the lysylbradykinin mutant had a significant improving effect on the symptoms of neurological deficit compared to the model group.

[0089] 2. Effect of the test substance on cerebral infarction size As shown in Table 9-1, the mutant PEG conjugates had a significant improving effect on cerebral infarction size compared to the model group. Considering the overall results of the neurological deficit score and cerebral infarction size, when the PEG conjugates were prepared using the same PEG modifier, the lysylbradykinin mutant-PEG conjugates showed superior efficacy to the lysylbradykinin-PEG conjugates.

[0090] [Table 15]

[0091] Looking at the data from Examples 7 to 10 overall, PEG conjugates of lysylbradykinin and its different mutants all showed significant pharmacological efficacy, and PEG conjugates of Lys-BK, mutants Lys-BKm01, Lys-BKm02, Lys-BKm11, Lys-BKm12, Lys-BKm13, Lys-BKm15, and Lys-BKm16 had significant improving effects on neurological deficit symptoms and cerebral infarction area compared to the model group, with statistically significant differences.

[0092] All of the lysylbradykinin / bradykinin mutants modified with PEG modifiers of different molecular weights (2K, 5K, 10K) showed good pharmacological efficacy. The Examples show the results of evaluating the in vivo activity of lysylbradykinin / bradykinin mutants modified with the modifying agent SPA. In fact, when lysylbradykinin / bradykinin or its mutants that retain their receptor binding activity are modified with other types of PEG modifiers, the half-life of lysylbradykinin / bradykinin in the body is significantly extended and good in vivo activity is shown.

[0093] On the other hand, non-PEG-modified lysylbradykinin / bradykinin or its different mutants (wild-type lysylbradykinin / bradykinin, m01, m02, m11, m12, etc.), or PEG conjugates of lysylbradykinin / bradykinin that do not retain receptor binding activity, when administered at different doses and through various routes of administration, did not show any effect in improving neurological deficit symptoms and cerebral infarction in animal models, which may be related to their very short half-lives in vivo.

[0094] Of course, there are some differences in the efficacy of different lysylbradykinin mutant-PEG conjugates. Among them, the PEG conjugate of the Lys-BKm13 mutant has the best efficacy. The Lys-BKm13 group showed a statistically significant improvement in neurological deficit symptoms and cerebral infarction size compared to the model group (P<0.001) (Table 7-4). Meanwhile, the Lys-BKm11 group showed a statistically significant improvement in neurological deficit symptoms and cerebral infarction size compared to the model group (P<0.01) (Table 7-3). Since Lys-BKm13 and Lys-BKm11 only differ in the first amino acid, it was suggested that mutating the first amino acid to Cys is significantly advantageous in in vivo efficacy.

[0095] Lys-BKm11 is a mutant obtained by inserting Arg between the first and second amino acids of Lys-BKm01, and has a certain advantage in improving cerebral infarction size (Table 7-3). This conclusion was further clarified by comparing the efficacy of Lys-BKm13 and Lys-BKm15. Under the same conditions, the PEG conjugate of the Lys-BKm13 mutant showed better efficacy ( Table 7-6). Furthermore, Lys-BKm16 was obtained by inserting another Arg based on Lys-BKm13. The efficacy of the PEG conjugate of Lys-BKm16 was equivalent to that of the PEG conjugate of Lys-BKm13 (Table 7-5). This indicates that the insertion of Arg at the N-terminus to extend the peptide is advantageous for exerting the efficacy of the peptide-PEG conjugate.

[0096] Lys-BKm01 is a mutant obtained by introducing a non-natural amino acid into Lys-BK by mutation. The PEG conjugate of Lys-BKm01 shows better efficacy than the PEG conjugate of Lys-BK (Table 9-1).

[0097] Lys-BKm06 is a mutant obtained by extending the C-terminus of Lys-BKm01. At a dose of 3 mg / kg, the PEG-modified Lys-BKm06 showed a tendency to improve neurological deficit symptoms and cerebral infarction size, but there was no significant difference compared to the model group, and the in vivo efficacy was lower than that of the PEG conjugate of Lys-BKm01 (Table 7-2). This indicates that the C-terminal structure is very important for the efficacy and activity of bradykinin. This is consistent with the results of confirming the minimum active unit in Example 3. When the dose was further increased, the PEG-modified Lys-BKm06 showed a significant improvement effect on neurological deficit symptoms and cerebral infarction size.

[0098] In summary, the present invention has the following advantages over the prior art: First, the present invention uses polyethylene glycol modification technology to significantly extend the half-life of lysylbradykinin or bradykinin in vivo, while at the same time exerting its biological activity well, solving the problem that the half-life of lysylbradykinin or bradykinin alone is too short and therefore not druggable. Regarding in vivo efficacy, PEG-modified lysylbradykinin or bradykinin and its variants showed a significant improvement effect on neurological deficit symptoms in MCAO cerebral ischemia-reperfusion model, and a significant improvement effect on cerebral infarction area.

[0099] Second, the modified lysylbradykinin or bradykinin mutants of the present invention have obvious advantages: they are significantly more stable in serum systems than the wild type while retaining their affinity for the receptor, suggesting a longer half-life in vivo, and after modification with PEG, they have better in vivo efficacy than the PEG-modified wild type lysylbradykinin or bradykinin.

[0100] Thirdly, the PEG-modified lysylbradykinin or bradykinin of the present invention can exert a remarkable medicinal effect even when administered by various administration routes such as intravenous injection, subcutaneous injection, oral administration, etc. In particular, subcutaneous injection and oral administration are convenient for clinical use because patients do not need to go to the hospital regularly to receive injections and can self-administer at home, effectively improving patient compliance, and are more suitable for long-term therapeutic administration such as the treatment of stroke patients in the recovery period.

Claims

1. A polyethylene glycol-modified lysylbradykinin or bradykinin, characterized in that the polyethylene glycol modification is an N-terminal modification and not a C-terminal modification, and the lysylbradykinin or bradykinin is a wild-type lysylbradykinin or bradykinin, or a derivative obtained by modifying the wild-type lysylbradykinin or bradykinin.

2. The polyethylene glycol-modified lysylbradykinin or bradykinin of claim 1, characterized in that the PEG-modified agent includes, but is not limited to, SPA, SCM, and SS-modified agents.

3. 2. The polyethylene glycol-modified lysylbradykinin or bradykinin according to claim 1, characterized in that the PEG modifier is a linear or branched PEG modifier.

4. 4. The polyethylene glycol-modified lysylbradykinin or bradykinin according to claim 3, characterized in that the PEG modifier is a linear PEG modifier.

5. The polyethylene glycol-modified lysylbradykinin or bradykinin according to claim 1, characterized in that the molecular weight of the PEG modifier is 2KD-20KD.

6. 2. The polyethylene glycol-modified lysylbradykinin or bradykinin according to claim 1, characterized in that the molecular weight of the PEG modifier is 2KD or 10KD.

7. A derivative of lysylbradykinin or bradykinin, characterized in that a cysteine ​​is introduced at any position in the amino acid sequence of wild-type lysylbradykinin or bradykinin.

8. 8. A derivative of lysylbradykinin or bradykinin according to claim 7, characterized in that Lys at position 1 of the amino acid sequence of wild-type lysylbradykinin is mutated to Cys.

9. A derivative of lysylbradykinin or bradykinin as described in claim 7, characterized in that any number and type of amino acids are inserted between the 1st and 2nd amino acid residues of the N-terminus of wild-type lysylbradykinin, or any number and type of amino acids are inserted before the 1st amino acid residue of the N-terminus of wild-type bradykinin.

10. A derivative of lysylbradykinin or bradykinin as described in claim 7, characterized in that one, two or three amino acids are inserted between the 1st and 2nd amino acid residues of the N-terminus of wild-type lysylbradykinin, or one, two or three amino acids are inserted before the 1st amino acid residue of the N-terminus of wild-type bradykinin.

11. The lysylbradykinin or bradykinin derivative according to any one of claims 9 and 10. A derivative of lysylbradykinin or bradykinin, characterized in that the inserted amino acid is arginine.

12. A lysylbradykinin or bradykinin derivative as described in claim 7, characterized in that it is composed of two or more lysylbradykinin molecules, or two or more bradykinin molecules, or a lysylbradykinin molecule and a bradykinin molecule linked together, and the lysylbradykinin or bradykinin molecule may be a wild-type lysylbradykinin or bradykinin, or a mutant obtained by substituting, inserting or deleting some amino acids based on the wild-type lysylbradykinin or bradykinin.

13. A derivative of lysylbradykinin or bradykinin as described in claim 7, characterized in that Phe at position 6 of wild-type lysylbradykinin or Phe at position 5 of wild-type bradykinin is mutated to any other amino acid.

14. A derivative of lysylbradykinin or bradykinin according to claim 7, characterized in that the sequence of said derivative is the sequence shown in SEQ ID NO:

14.

15. A polyethylene glycol-modified lysylbradykinin or bradykinin according to any one of claims 1 to 6, characterized in that the lysylbradykinin or bradykinin is a derivative of the lysylbradykinin or bradykinin according to any one of claims 7 to 14.

16. The polyethylene glycol-modified lysylbradykinin or bradykinin according to any one of claims 1 to 6 and 15 for the treatment, prevention, improvement of prognosis, or prevention of recurrence of ischemic stroke.

17. The polyethylene glycol-modified lysylbradykinin or bradykinin according to any one of claims 1 to 6 and 15, which is administered by injection or orally.

18. The lysylbradykinin or bradykinin derivative according to any one of claims 7 to 13 for treating, preventing, improving the prognosis, or preventing recurrence of ischemic stroke.

19. The lysylbradykinin or bradykinin derivative according to any one of claims 7 to 13, which is administered by injection or orally.

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