Application of kinin or derivative thereof in treatment of hypertension and prevention or treatment of hypertension complications

Abstract

The provided long-acting kinin or the derivative thereof fundamentally solves the problem that kinin has no druggability, on one hand, the half-life period of kinin or the derivative thereof is prolonged through a long-acting technology, and it is prevented that kinin or the derivative thereof is rapidly degraded and inactivated in vivo; on the other hand, the action site of the kinin and the receptor is covered or partially covered through long-acting modification, so that the adverse reactions such as rapid blood pressure reduction and hypotension in a short time are effectively avoided. The long-acting kinin or the derivative thereof well retains receptor binding activity and signal transduction activity in an in-vitro cell experiment, can effectively treat hypertension and prevent or treat hypertension complications in an in-vivo experiment, and provides a new method for treating hypertension and hypertension complications.

Description

Application of kinin or its derivatives in treating hypertension, preventing or treating complications of hypertension Technical Field

[0001] The present invention relates to kinin or its derivatives in the field of biomedicine, in particular to the use of long-acting kinin or its derivatives in treating hypertension and preventing or treating complications of hypertension. Background Art

[0002] Hypertension is a leading cause of premature death and cardiovascular disease worldwide. The prevalence of hypertension among adults in my country is approximately 27.5%. In recent years, awareness, treatment, and control rates among hypertension patients in my country have achieved significant results, reaching 51.6%, 45.8%, and 16.8%, respectively. However, overall, these rates remain relatively low.

[0003] Hypertension refers to a clinical syndrome characterized by increased systemic arterial blood pressure (systolic and / or diastolic blood pressure) (the 2022 "Chinese Clinical Practice Guidelines for Hypertension" recommends adjusting the diagnostic threshold for hypertension in adults in my country to systolic blood pressure greater than or equal to 130 mmHg and / or diastolic blood pressure greater than or equal to 80 mmHg), which may be accompanied by functional or organic damage to organs such as the heart, brain, and kidneys. At the same time, hypertension is also a major risk factor for many diseases. Hypertension can cause coronary artery lesions, leading to insufficient blood supply to myocardial cells, and ultimately leading to complications such as coronary heart disease, angina pectoris, and even myocardial infarction; hypertension can cause cerebral vascular lesions, and insufficient blood supply to brain tissue can cause brain cell death and induce complications such as ischemic cerebral infarction, vascular cognitive impairment, and cerebral small vessel disease; hypertension can also cause renal artery lesions and ocular capillary lesions, leading to hypertensive nephropathy and hypertensive eye disease.

[0004] Commonly used antihypertensive drugs in clinical practice still have many shortcomings in lowering blood pressure and improving symptoms. Modern medicine still has many unknowns about the exact cause and pathogenesis of hypertension, and the development of more effective treatments is extremely necessary.

[0005] Summary of the Invention

[0006] The first technical problem solved by the present application is to provide a new drug for treating hypertension, and specifically to provide the use of kinin or its derivatives in the preparation of a drug for treating or improving hypertension.

[0007] The present invention also provides a pharmaceutical composition for treating or improving hypertension, comprising kinin or a derivative thereof.

[0008] The present invention also provides a method for treating or improving hypertension, which comprises administering kinin or its derivatives to a patient.

[0009] The second technical problem solved by the present application is to provide a new drug for preventing or treating complications of hypertension. Specifically, it provides the use of kinin or its derivatives in the preparation of a drug for preventing or treating complications of hypertension.

[0010] The present application also provides a pharmaceutical composition for preventing or treating complications of hypertension, comprising kinin or its derivatives.

[0011] The present invention also provides a method for preventing or treating complications of hypertension, comprising administering kinin or its derivatives to a patient.

[0012] The complications of hypertension include hypertensive heart and vascular diseases, hypertensive encephalopathy, hypertensive nephropathy, etc.

[0013] The above-mentioned kinins include bradykinin or lysyl bradykinin.

[0014] The above-mentioned kinin or its derivative is a long-acting kinin or its derivative.

[0015] The kinin or its derivatives can be administered alone or in combination with other drugs for resisting hypertension or preventing or treating complications of hypertension.

[0016] The administration method includes, but is not limited to, injection, oral administration, etc. The injection administration method includes, but is not limited to, intravenous injection, subcutaneous injection, etc.

[0017] Peptide drugs generally suffer from poor stability and a short half-life in vivo, especially those used to treat chronic diseases, which often require long-term, frequent administration. Lysyl bradykinin / bradykinin is degraded in vivo primarily by aminopeptidases, carboxypeptidases, and angiotensin ACEII. Bradykinin's dynamic equilibrium is achieved through a system of kallikreins and degrading enzymes. Compared to common peptides, lysyl bradykinin and bradykinin are more unstable, with reported half-lives of only seconds. Exogenously administered bradykinins are rapidly hydrolyzed and inactivated by kininase in vivo.

[0018] In order to improve the stability of peptide drugs, prolong their half-life in vivo, and make them long-acting, two main methods are currently used: 1. Molecular modification of peptides, usually using strategies such as amino acid substitution or cyclization, PEG modification, fusion of long-acting fragments (fusion with Fc fragments, fusion with human serum albumin), and conjugation with fatty acids; 2. Long-acting peptide drugs through formulation means.

[0019] In the specific examples of this application, two methods are used to extend the half-life of kinin, namely, polyethylene glycol modification of kinin and optimization of the amino acid sequence of kinin.

[0020] At the polyethylene glycol modification level, the polyethylene glycol-modified lysyl bradykinin or bradykinin provided by the present invention, the PEG modification is an N-terminal modification, not a C-terminal modification, and the lysyl bradykinin or bradykinin is a wild-type lysyl bradykinin or bradykinin, or a derivative obtained by transformation (non-polyethylene glycol modification) based on the wild-type lysyl bradykinin or bradykinin. The derivatives include not only the various mutants exemplified in the examples of the present application, but also polypeptides, fusion proteins (including but not limited to albumin fusion, Fc fusion, etc.) and various forms of modifications (except polyethylene glycol modification) obtained by further transformation based on the Lys-BK / BK mutants of the present application, and the derivatives retain the in vitro activity (binding activity to the receptor) of the wild-type lysyl bradykinin or bradykinin. The embodiment exemplifies the stability, in vitro activity, and in vivo drug efficacy of the PEG modifications of lysyl bradykinin or bradykinin and its various variants, and the results all show good stability, and the lysyl bradykinin or bradykinin variants having in vitro activity can all be solved after being modified with polyethylene glycol to extend the half-life of lysyl bradykinin or bradykinin and to exert its in vivo biological activity; On the contrary, other lysyl bradykinins or bradykinin variants that do not retain in vitro activity are modified with polyethylene glycol, or lysyl bradykinin or bradykinin and its various variants (without PEG modification) have no drug efficacy in vivo. Therefore, those skilled in the art can reasonably expect that other lysyl bradykinins or bradykinin derivatives having retained receptor binding activity can all be solved after being modified with polyethylene glycol to extend the half-life of lysyl bradykinin or bradykinin and to exert its in vivo biological activity, and are not limited to the specific lysyl bradykinin or bradykinin variants exemplified in the embodiment. The present application uses PEG modification technology to significantly prolong the half-life of lysyl bradykinin or bradykinin while exerting its biological activity in vivo.

[0021] In specific embodiments of the present application, the modifiers used include SPA, SCM, and SS modifiers; and can be linear or branched PEG modifiers.

[0022] Preferably, the PEG modifier is a linear PEG modifier.

[0023] Preferably, the molecular weight of the PEG modifier is 2KD-20KD.

[0024] Preferably, the molecular weight of the PEG modifier is 5KD-10KD.

[0025] Lysyl bradykinin or bradykinin act on corresponding receptors and play a series of physiological effects. However, the half-life of lysyl bradykinin and bradykinin in vivo is extremely short, only a few seconds, and they are quickly hydrolyzed and inactivated by kininase, and their short biological half-life greatly limits their drugability. The application, by polyethylene glycol modification technology, greatly prolongs the half-life of lysyl bradykinin or bradykinin in vivo, while playing its biological activity in vitro and in vivo well, and produces significant in vivo activity in spontaneously hypertensive rat models, solving the problem that simple lysyl bradykinin or bradykinin half-life is extremely short and cannot be drugged.

[0026] At the level of optimizing the amino acid sequence of kinin, the kinin mutants of the present application retain the in vitro activity (i.e., receptor binding activity) of wild-type lysyl-bradykinin or bradykinin, and include not only the various mutants listed in the examples of the present application, but also polypeptides and fusion proteins (including but not limited to albumin fusion, Fc fusion, etc.) obtained by further modification based on the Lys-BK / BK mutants of the present application.

[0027] In a specific embodiment of the present application, cysteine ​​(Cys) is introduced at any position in the wild-type lysyl bradykinin or bradykinin sequence. Preferably, the first Lys of the wild-type lysyl bradykinin is mutated to Cys, i.e., CRPPGFSPFR, the sequence of which is shown in SEQ ID NO: 18.

[0028] In a specific embodiment of the present application, any number and type of amino acids are inserted between the first and second amino acid residues at the N-terminus of the wild-type lysyl bradykinin, i.e., KXRPPGFSPFR, the sequence of which is shown in SEQ ID NO: 19, where X represents any number and any type of amino acids. Any amino acid is inserted before the first amino acid residue at the N-terminus of the wild-type bradykinin, i.e., XRPPGFSPFR, the sequence of which is shown in SEQ ID NO: 20, where X represents any number and any type of amino acids. Experimental results show that the activity is better after the N-terminus is extended. Preferably, one, two, or three amino acids are inserted. Preferably, one or two arginines (Arg) are inserted.

[0029] In a specific embodiment of the present application, a lysyl bradykinin or bradykinin derivative is composed of two or more lysyl bradykinin monomers connected in series (such as m14), or two or more bradykinin monomers connected in series, or a lysyl bradykinin monomer and a bradykinin monomer connected in series (such as m12). The lysyl bradykinin or bradykinin monomer can be a wild-type lysyl bradykinin or bradykinin, or it can be a variant in which some amino acids are replaced, inserted or deleted on the basis of the wild-type lysyl bradykinin or bradykinin.

[0030] In a specific embodiment of the present application, the Phe at position 6 of the wild-type lysyl bradykinin or the Phe at position 5 of the wild-type bradykinin is mutated to other amino acids, such as the unnatural amino acids Igl (α-2-indolylglycine) and THI (β-2-thiophenylalanine). The LBK or BK mutated at these sites is less easily recognized by enzymes in the body, thereby effectively extending its half-life in the body.

[0031] Preferably, the lysyl bradykinin or bradykinin derivative of the present application has a sequence as shown in SEQ ID NO: 14.

[0032] The polyethylene glycol modification method and amino acid sequence optimization used in the specific examples of this application are only used to illustrate that the half-life of kinin can be extended by the above methods, and are not limited to the specific implementation methods of extending the half-life of kinin. Those skilled in the art can also use other polyethylene glycol modification methods and other amino acid sequence optimization methods other than those exemplified in this application, or use other modification methods for long-acting polypeptide drugs (such as fusion with Fc fragment, fusion with human serum albumin, conjugation with fatty acids, and formulations as described above) to modify kinin to make it long-acting.

[0033] Spontaneously hypertensive rats (SHRs) are the preferred animal model for screening antihypertensive drugs. While normal rats have a systolic blood pressure of 110-120 mmHg, SHRs can reach peak blood pressures of 180-200 mmHg after development.

[0034] After birth, SHR blood pressure increases with age. As the disease progresses, SHR can develop complications such as heart, brain, and kidney disease, as well as vascular lesions. Therefore, in addition to its application in drug screening for hypertension, the SHR model is increasingly being used to study other diseases.

[0035] Left ventricular hypertrophy and myocardial fibrosis are common complications of hypertension and independent risk factors for arrhythmias, heart failure, and sudden death. Early blood pressure control and the prevention and reversal of left ventricular hypertrophy have become key priorities in the prevention and treatment of hypertension. SHRs (Serious Hypertensive Respiratory Syndrome) exhibit hypertensive cardiac and vascular pathology. Multiple studies have confirmed that SHRs are an ideal animal model for studying hypertensive left ventricular hypertrophy and heart failure, and are therefore suitable for studying cardiac dysfunction caused by hypertension.

[0036] SHR is also a model of hypertensive brain damage. The time-dependent increase in SHR arterial blood pressure, brain atrophy, and nerve cell loss are similar to the occurrence of human hypertensive encephalopathy to a certain extent.

[0037] In addition, long-term hypertension can lead to renal arteriosclerosis, proteinuria, decreased renal function, and in severe cases, renal failure. Several studies have confirmed that SHR is an ideal model for studying hypertensive nephropathy.

[0038] The results of this study show that kinin or its derivatives can significantly lower blood pressure and significantly improve heart, brain, and kidney function in SHR after administration. Therefore, kinin or its derivatives can be used to treat hypertension and prevent hypertension complications.

[0039] The kallikrein-kinin system, or KKS, is involved in a variety of physiological and pathological processes, including the regulation of cardiovascular, renal, and nervous system function. The KKS comprises kallikrein (KLK), kininogens, kinins, kinin receptors (B1 and B2 receptors), and kininase. Kallikrein, also known as kininogenases or kallikreins, is a serine protease classified into two major classes: plasma kininogenases (PK) and tissue kininogenases (TK), both of which play crucial physiological roles. Human tissue kininogenases are believed to consist of at least 15 members (KLK1-KLK15), with tissue kininogenase 1 (KLK1) being the most studied. KLK1 converts kininogens into kinins, which act on their corresponding receptors to exert a range of biological effects. The best-studied kinins include lysyl-bradykinin (a decapeptide) and bradykinin (a nonapeptide). Lysyl-bradykinin is cleaved into bradykinin by aminopeptidases in vivo. Both lysyl-bradykinin and bradykinin (a nonapeptide) have diverse physiological functions, including regulating blood pressure and inflammation. Although bradykinin / lysyl bradykinin has the physiological effects of vasodilation and lowering blood pressure, their half-life in the body is very short, only a few seconds to a few minutes. Exogenous bradykinin / lysyl bradykinin will be rapidly degraded when it enters the body; at the same time, large doses of exogenous bradykinin / lysyl bradykinin entering the body will cause a rapid drop in blood pressure and adverse reactions such as hypotension. These greatly limit their drugability, and there are currently no kinin drugs on the market. The present application found that long-acting kinin or its derivatives can fundamentally solve the problem of bradykinin / lysyl bradykinin being non-drugable. On the one hand, the long-acting technology increases the half-life of kinin or its derivatives, preventing it from being rapidly degraded and inactivated in the body; on the other hand, the long-acting modification technology covers or partially covers the kinin and receptor action sites, effectively avoiding the occurrence of rapid blood pressure drops, hypotension and other adverse reactions in a short period of time. The long-acting kinin or its derivatives of the present application are used to treat hypertension and prevent hypertensive complications, providing a new method for treating hypertension and preventing hypertensive complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1: Lys-BK and its variants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins. β-actin is an internal control among the five signals; p- represents the phosphorylation of the corresponding protein; control is a buffer blank control; Lys-BK-M06-EH is a sample after incubation with serum mixed with M06.

[0041] Figure 2: Detection of the binding activity of Lys-BK and its variant PEG conjugates to B2 receptor (reporter gene method)

[0042] Figure 3: Lys-BK and its variant PEG conjugates activate phosphorylation of downstream ERK1 / 2 and CREB proteins. β-actin is an internal reference among the five signals; p- represents phosphorylation of the corresponding protein; control is a buffer blank control.

[0043] Figure 4: Effects of the test drug in Example 6 on blood pressure in spontaneously hypertensive rats

[0044] Figure 5: Effects of the test drug in Example 7 on blood pressure in spontaneously hypertensive rats

[0045] Figure 6: Effects of the test drug in Example 8 on blood pressure in spontaneously hypertensive rats

[0046] Figure 7: Effects of the test drug in Example 8 on the volume fraction of collagen in the aorta media of spontaneously hypertensive rats (Masson staining ×400), where A is the negative control group and B is the Lys-BKm13-SPA10K group

[0047] Figure 8: Effects of the test drug in Example 8 on the volume fraction of elastic fibers in the aorta media of spontaneously hypertensive rats (Gomori aldehyde fuchsin staining ×400), where A is the negative control group and B is the Lys-BKm13-SPA10K group

[0048] Figure 9: Effects of the test drug of Example 9 on blood pressure in spontaneously hypertensive rats after 4 weeks of administration

[0049] Figure 10: Effects of the test drug of Example 9 on blood pressure in spontaneously hypertensive rats after 8 weeks of administration

[0050] Figure 11: Effects of the test drug of Example 10 on blood pressure in spontaneously hypertensive rats after 4 weeks of administration

[0051] Figure 12: Effects of the test drug of Example 10 on blood pressure in spontaneously hypertensive rats after 8 weeks of administration DETAILED DESCRIPTION

[0052] Unless otherwise specified, the technical terms or abbreviations used in this application have the following meanings:

[0053] [Corrected 06.09.2024 according to Rule 91] Lys-BK: wild-type lysyl-bradykinin, which is consistent with the sequence of natural human lysyl-bradykinin; the amino acid sequence is KRPPGFSPFR, as shown in SEQ ID NO: 1.

[0054] [Corrected 06.09.2024 according to Rule 91] BK: wild-type bradykinin, which is consistent with the sequence of natural human bradykinin; the amino acid sequence is RPPGFSPFR, as shown in SEQ ID NO: 21.

[0055] Mutant: A variant obtained by replacing, inserting or deleting some amino acids based on wild-type lysyl-bradykinin or bradykinin.

[0056] Derivatives include not only the various mutants listed in the examples of this application, but also polypeptides, fusion proteins (including but not limited to albumin fusion, Fc fusion, etc.), and various forms of modifications obtained by further modification based on the Lys-BK / BK mutants of this application.

[0057] Polyethylene glycol: PEG, usually produced by the polymerization of ethylene oxide, is available in branched, linear, and multi-arm forms. Ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group, methoxy polyethylene glycol (mPEG) is obtained.

[0058] Polyethylene glycol modifiers: PEG modifiers are functionalized polyethylene glycol derivatives that are activated polyethylene glycols and can be used to modify proteins and peptides. The polyethylene glycol modifiers used in this application were purchased from Jiangsu Zhonghong Bioengineering Pharmaceutical Research Institute Co., Ltd. or Beijing Jiankai Technology Co., Ltd. The actual molecular weight of a PEG modifier with a specific molecular weight can be 90% to 110% of the listed value. For example, the molecular weight of PEG5K can be 4.5 kDa to 5.5 kDa.

[0059] M-SPA-5K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 5 kDa, the structure of which is shown in formula (1), where n is an integer from 98 to 120.

[0060] M-SPA-2K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 2 kDa, the structure of which is shown in formula (1), where n is an integer from 37 to 45.

[0061] M-SPA-10K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 10 kDa, the structure of which is shown in formula (1), where n is an integer from 200 to 245.

[0062] M-SPA-20K: linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of approximately 20 kDa, the structure of which is shown in formula (1), where n is an integer from 405 to 495.

[0063] M-SCM-5K: linear monomethoxy polyethylene glycol succinimidyl acetate with a molecular weight of about 5 kDa, the structure of which is shown in formula (2), where n is an integer from 99 to 120.

[0064] M-SS-5K: linear monomethoxy polyethylene glycol succinimidyl succinate with a molecular weight of about 5 kDa, the structure of which is shown in formula (3), where n is an integer from 98 to 119.

[0065] M-YNHS-10K: branched monomethoxy polyethylene glycol succinimidyl acetate with a molecular weight of about 10 kDa, the structure of which is shown in formula (4), where n is an integer from 98 to 120.

[0066] Example 1 Design and Synthesis of Lysyl-Bradykinin / Bradykinin Variants

[0067] Based on the sequence of lysyl bradykinin / bradykinin and its potential degradation pathways in vivo, a series of variants were designed (Table 1-1). These variants primarily consisted of substitutions with structurally similar natural and unnatural amino acids (m01-m04, m07, with m02 being a BK variant). To identify the minimum active unit for biological activity / function, partially truncated or extended peptide variants were designed (m05, m06, m08-m11). Cysteines were introduced (m13, m15, and m16) to further enhance the antioxidant properties of the variants. To further improve peptide stability, lysyl bradykinin / bradykinin monomers were tandemly connected (m12 is a Lys-BK tandem with BK, and m14 is two Lys-BK tandems). Based on the m13 peptide, the effects of N-terminal extension on efficacy were compared, and variants m15 and m16 were designed. All peptides achieved purity >95%.

[0068] Table 1-1 Lysyl bradykinin / bradykinin variant sequences

[0069] Example 2 Study on the stability of lysyl bradykinin / bradykinin and its variants

[0070] The stability of lysyl bradykinin / bradykinin was evaluated by evaluating the stability in an in vitro serum system. 2 mg / ml peptide and SD rat serum were mixed at a volume ratio of 85:15 and incubated at 37°C for 30 min, 60 min, 90 min, 120 min, 180 min and other time points. The reaction was terminated by adding 3% trichloroacetic acid (50% volume of the above peptide serum mixture system) and the matrix interference in the serum was effectively removed. Further based on UPLC-RP-UV 214nm Methods The stability of peptide samples was evaluated by monitoring their degradation in serum. RP analysis method: chromatographic column Protein BEH C4 2.1x150mm, 1.7μm, column temperature 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.

[0071] Table 2-1 Stability of lysyl bradykinin and its variants in serum system

[0072] The results in Table 2-1 show that the designed polypeptide variants are more stable in serum than the wild-type lysyl bradykinin, which also indicates that they have certain advantages in stability and efficacy in vivo.

[0073] Example 3 Evaluation of in vitro activity of lysyl-bradykinin / bradykinin and its variants

[0074] 1. B2 receptor binding activity based on reporter gene assay

[0075] The in vivo pharmacological effects of lysyl-bradykinin / bradykinin are achieved by binding to the B2 receptor and activating downstream signaling pathways. A B2 receptor reporter gene stably transfected cell line B2-NFAT-CHO-K1 was constructed based on CHO-K1 host cells and a cell bank was established. This cell line was used to detect and evaluate the in vitro binding activity of lysyl-bradykinin / bradykinin or its variants, as well as PEG-modified products, to the B2 receptor.

[0076] Detection method: cells were plated at 5×10 4 The cells were plated at a density of 1000 cells / well in a 96-well plate and cultured at 37°C, 5% CO2 for 16-20 hours. The standard and test solutions were then diluted in series. After 5 hours, the Bio-Lite luciferase assay reagent was added to measure the chemiluminescence intensity. GraphPad was used to plot the curve and the four-parameter equation was fitted according to the EC curve. 50 The relative activity was calculated based on the values. Table 3-1 lists some of the test results. The relative binding activities of the peptide variants to the B2 receptor are listed. Among them, the activities of Lys-BKm11, Lys-BKm13, Lys-BKm15 and Lys-BKm16 variants are significantly improved compared with the wild-type lysyl bradykinin.

[0077] Table 3-1 Relative binding activity of Lys-BK and its variants to B2 receptor

[0078] 2. Activity analysis of key target proteins in downstream signaling pathways

[0079] Using B2-NFAT-CHO-K1 cells overexpressing B2 receptors, we treated cells with Lys-BK / BK or its variants in vitro. Western blotting was used to assess changes in p-CREB and p-ERK1 / 2, downstream B2 receptor signaling molecules (P indicates phosphorylation; phosphorylation of serine or threonine residues activates proteins, leading to a series of effects within the pathway). These changes were used to compare the activity of the test compounds. Extracellular signal-regulated protein kinases 1 / 2 (ERK1 / 2) and cAMP-response element binding protein (CREB) are both direct downstream signaling molecules of B2 receptor activation. Activation of CREB and ERK1 / 2 after stroke can prevent post-ischemic inflammation and neuronal damage. Therefore, their expression levels can, to a certain extent, reflect the ability of the test compound to activate B2 receptors and, in turn, the significance of its neuroprotective effects.

[0080] CHO-K1 (B2R+) cells in the logarithmic growth phase were collected and the cell population was adjusted to 3×10 6 cells / mL, seeded in cell culture plates, 3×10 5 Cells were cultured in a 37.0°C, 5.0% CO2 incubator for 24-48 hours. When cell density reached 90% or higher, the cells were washed with PBS and 1 ml of 1 μM Lys-BK / BK or its variant was added to each well. A control group was treated with F12 medium. The cell culture plate was incubated in a 37.0°C, 5.0% CO2 incubator for 5 minutes, followed by washing with PBS. 80 μL of protein lysis buffer (RIPA, PMSF, phosphatase inhibitors) was added to each well, and the bottom of the wells were scraped back and forth with a scraper to accelerate cell lysis. The cell fragments and lysis buffer were transferred to a centrifuge tube and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was aspirated and mixed with 5× Loading Buffer at a 4:1 volume ratio. The supernatant was then boiled in a 100°C metal bath for 10 minutes. Equal amounts of protein samples were loaded onto a 10% SDS-PAGE gel for running, transfer to a membrane, blocking, antibody incubation, and luminescence analysis.

[0081] The results of the assay for activation of downstream signaling by Lys-BK / BK or its variants are shown in Figure 1. Lysyl-bradykinin (Lys-BK) can activate phosphorylation of downstream ERK1 / 2 and CREB proteins, consistent with the drug's mechanism of action. The in vitro biological activity of the samples was qualitatively evaluated by assessing the phosphorylation levels of downstream ERK1 / 2 and CREB proteins. The evaluation results showed that the Lys-BKm05 and Lys-BKm07 variants had lost their downstream pathway activation activity, while LBKm01, m02, m06, m11, m12, m13, m14, m15, and m16 all displayed downstream pathway activation activity.

[0082] [Corrected 06 / 09 / 2024 according to Rule 91] Based on the results of the two in vitro activity evaluations described above, it was shown that truncation of the C-terminus of lysyl-bradykinin / bradykinin lost its B2R binding and signaling activity. Deletion of the N-terminal lysine of lysyl-bradykinin did not affect its B2R binding and signaling activity, while deletion of other amino acids at the N-terminus lost its B2R binding and signaling activity. The minimum active unit is RPPGFSPFR, as shown in SEQ ID NO: 21. N-terminal extension (m11) or introduction of a cysteine ​​(m13) both enhanced B2R binding activity. Figure 1d shows that CREB protein phosphorylation in the m12, 13, and 14 groups was significantly higher than that in the LBK group, indicating that m12, 13, and 14 groups can better activate downstream pathways and exhibit superior in vitro activity to LBK.

[0083] Example 4 Preparation of PEG Conjugates of Lysyl-Bradykinin / Bradykinin and Its Variants

[0084] PEGylation of lysyl bradykinin / bradykinin and its variants is performed using conventional methods in the art. Depending on the specific variant and PEG modifier, some parameters may vary slightly. These parameters can be optimized by those skilled in the art through limited experimentation, and there are no technical obstacles. Taking M-SPA-5K modified wild-type BK as an example, the preparation process is as follows:

[0085] 1. Preparation of PEG Conjugate Samples

[0086] Dissolve the peptide sample in sodium dihydrogen phosphate / disodium hydrogen phosphate buffer (pH 7.0) to a final concentration of 10 mg / mL. Add PEG to the peptide solution at a peptide to M-SPA-5K mass ratio of 1:7. Slowly stir until well mixed and incubate at 2-8°C for 1 hour.

[0087] 2. Purification of the reaction mixture

[0088] The first step of purification chromatography conditions are as follows:

[0089] The purification filler was GE's Superdex75 medium, and the purification mobile phase was pH 7.0-7.5, 20 mM sodium dihydrogen phosphate / disodium hydrogen phosphate buffer containing 0.2 M NaCl.

[0090] Sample loading: directly load the above peptide-PEG modification reaction mixture after the reaction is completed.

[0091] Elution: After loading, wash the column with mobile phase for at least 1.5 column volumes. 214nm Trend: Collect elution samples in steps.

[0092] The second step purification chromatography conditions are as follows:

[0093] The C18 reverse phase column separation and purification method achieves separation based on the difference in polarity between the two. The product purification yield can reach more than 80%, and free PEG can be completely removed.

[0094] 3. Purity Analysis of PEG Conjugate Samples

[0095] (1) HPLC purity analysis

[0096] The detection was carried out with reference to the general chapter 0512 high performance liquid chromatography of the 2020 edition of the "Chinese Pharmacopoeia". The detection chromatography type was SEC (molecular sieve chromatography), the mobile phase was 20mM PB7.0+10% IPA, the chromatographic column was TSK G2000, and the collection conditions were 214nm.

[0097] RP purity analysis method: chromatographic column Protein BEH C4 2.1x150mm, 1.7μm, mobile phase A: 0.1% TFA / H2O, mobile phase B: 0.1% TFA / ACN.

[0098] The purity results showed that the prepared lysyl bradykinin variant series PEG-modified products had uniform peaks, no obvious impurity peaks were observed, and the purity was >95%.

[0099] (2) SDS-PAGE purity analysis

[0100] The sample purity was determined according to the fifth method of electrophoresis, SDS-polyacrylamide gel electrophoresis, in the 2020 edition of the Chinese Pharmacopoeia 0541, and 12.5% ​​SDS-PAGE was used for sample testing.

[0101] The electrophoresis results showed that the prepared lysyl bradykinin variants had uniform bands with no mixed bands and a purity of >95%.

[0102] In addition, lysyl bradykinin / bradykinin or its variants have only the N-terminus with a theoretically possible modification site, so their PEG modifications are all N-terminal single-modification products.

[0103] Example 5 In vitro evaluation of PEG conjugates of lysyl-bradykinin / bradykinin and its variants

[0104] 1. Stability

[0105] The stability of the PEG conjugates of lysyl bradykinin and its variants was indirectly assessed by evaluating their stability in vitro in a serum system, and the detection method was as described in Example 2. The residual time of the PEG conjugates of lysyl bradykinin and its variants in an in vitro serum system was greater than 6h (Table 5-1), and the stability was significantly improved. Under the same molecular weight PEG, the stability of the branched conjugate Lys-BKm01-YNHS10K was better than that of the linear conjugate Lys-BKm01-SPA10K.

[0106] Table 5-1 Stability of PEG conjugates of lysyl bradykinin and its variants in serum system

[0107] 2. In vitro activity

[0108] The PEG conjugates of lysyl bradykinin and its variants have carried out in vitro binding activity and signal transduction activity with B2 receptors, and the detection method is the same as in Example 3. Lysyl bradykinin or its variants still have the binding activity (Fig. 2a, 2b) with B2 receptor after PEG modification, and can still activate the phosphorylation (Fig. 3) of B2 receptor downstream signaling pathway key protein. Different structural types, different molecular weight PEG modifier coupling products all still preferably retain the binding activity with B2 receptor, and PEG modifier includes: molecular weight from 2K to 20K, straight chain and branched, different structures (SPA, SCM, SS etc.). The result shows that under the same molecular weight, straight chain PEG conjugate activity is higher than branched chain PEG conjugate, and this result is all established on 10K and 20K two kinds of molecular weight PEG. As shown in Figure 2a, the EC50 values ​​of Lys-BKm01-SPA10K and Lys-BKm01-YNHS10K were 90.8 μg / ml and 346.6 μg / ml, respectively. The linear PEG conjugate exhibited nearly four times higher activity than the branched PEG conjugate. Furthermore, among the four PEG structures of the same type, 2K, 5K, 10K, and 20K, the 2K and 10K PEG conjugates exhibited relatively higher activity.

[0109] Results of downstream signaling pathway activation by PEG conjugates of lysyl-bradykinin and its variants (Figure 3) show that the peptides retained their activity in activating ERK1 / 2 and CREB phosphorylation downstream of the B2 receptor after PEG conjugation, although this activity was somewhat reduced compared to the Lys-BK peptide, particularly for ERK1 / 2 phosphorylation. Under the same SPA5K-PEG modifier, Lys-BKm13-SPA5K exhibited the highest activity among the different peptide variants. Under the same peptide variant, Lys-BKm01, 2K and 10K PEG-modified conjugates exhibited the highest activity. Furthermore, the linear PEG conjugate, Lys-BKm01-SPA10K, exhibited higher activity than the branched PEG conjugate, Lys-BKm01-YNHS10K.

[0110] In summary, the stability of PEG conjugates of lysyl bradykinin and its variants is significantly improved, and they can still well retain the B2 receptor binding activity and signal transduction activity before PEG conjugation.

[0111] Example 6 Comparison of the blood pressure lowering effect of long-acting BK in spontaneously hypertensive rats (1)

[0112] In the present invention, spontaneously hypertensive rats (SHR) were used as model animals to compare the blood pressure-lowering effects of a series of drugs administered via different routes (subcutaneous and oral).

[0113] 1. Grouping and Experimental Design

[0114] SHR rats were used as model animals, with a total of 100 male SHR rats, 16-18 weeks of age. The experiment was divided into four groups: a negative control group, a Lys-BKm13-SPA10K group (the test drug was a PEG conjugate of a kinin, the kinin being Lys-BKm13, modified with M-SPA-10K), a Lys-BKm13-SPA5K subcutaneous group (the test drug was a PEG conjugate of a kinin, the kinin being Lys-BKm13, modified with M-SPA-5K), and a valsartan group. There were 30 animals in each of the negative control group, the Lys-BKm13-SPA10K oral group, and the Lys-BKm13-SPA5K subcutaneous group, and 10 animals in the valsartan group. The drug was administered five times a week for a total of 8 weeks. Systolic blood pressure, diastolic blood pressure, and mean arterial pressure were measured the next day after completing 8 weeks of administration.

[0115] Table 6-1 Note: a: number of animals.

[0116] 2. Results

[0117] Hypertension in SHR rats is established after 16 weeks of age, with blood pressure reaching its highest level at 24 weeks. 16-24 weeks of age corresponds to 18-25 years in humans, or to a patient with initial hypertension. Compared to the negative control group, the PEG-conjugated kinin and valsartan groups showed significant or extremely significant decreases in blood pressure. See the table below for details.

[0118] Table 6-2 Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0119] Example 7 Comparison of the blood pressure lowering effect of long-acting BK in spontaneously hypertensive rats (2)

[0120] In the present invention, spontaneously hypertensive rats (SHR) were used as model animals to compare the blood pressure-lowering effects of different candidate molecular drugs on SHR rats after oral administration.

[0121] 1. Grouping and Experimental Design

[0122] SHR rats were used as model animals, with a total of 100 male SHR rats, aged 30 to 32 weeks. There were 4 groups in the experiment, namely the negative control group, the Lys-BKm13-SPA5K group (the test drug was a PEG conjugate of kinin, the kinin was Lys-BKm13, modified with M-SPA-5K), the Lys-BKm13-SPA10K group (the test drug was a PEG conjugate of kinin, the kinin was Lys-BKm13, modified with M-SPA-10K), and the valsartan group. There were 30 animals in each of the negative control group, the Lys-BKm13-SPA5K group, and the Lys-BKm13-SPA10K group, and 10 animals in the valsartan group. The drug was administered 5 times a week for a total of 4 weeks. The systolic blood pressure, diastolic blood pressure, and mean arterial pressure of all animals were tested the next day after completing the 4-week drug administration. The grouping and drug administration information are as follows:

[0123] Table 7-1 Note: a: number of animals.

[0124] 2. Results

[0125] Hypertension in SHR rats is established after 16 weeks of age, with blood pressure reaching its highest level at 24 weeks. Ages 30 to 34 weeks correspond to 25 to 30 years in humans, or equivalent to a patient with hypertension for 8 to 12 years. Compared to the negative control group, the PEG conjugates of kinin and valsartan groups showed significant or extremely significant decreases in blood pressure. See the table below for details.

[0126] Table 7-2 Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0127] Example 8 Comparison of the blood pressure lowering effect of long-acting BK in spontaneously hypertensive rats (3)

[0128] In the present invention, spontaneously hypertensive rats (SHR) were used as model animals to compare the blood pressure-lowering effects of different doses on SHR rats.

[0129] 1. Grouping and Experimental Design

[0130] SHR rats were used as model animals, with a total of 100 male SHR rats, aged 40-42 weeks. The experiment consisted of four groups: a negative control group, a Lys-BKm13-SPA10K group, and a valsartan group. There were 30 animals in each of the negative control group and the Lys-BKm13-SPA10K group, and 10 animals in the valsartan group. The drug was administered five times a week for a total of four weeks. The systolic blood pressure, diastolic blood pressure, and mean arterial pressure of all animals were measured the next day after the four-week administration. The grouping and dosing information are shown in the following table:

[0131] Table 8-1 Note: a: number of animals.

[0132] 2. Results

[0133] Hypertension is established in SHR rats after 16 weeks of age, with blood pressure reaching its highest level at 24 weeks. Ages 40-44 weeks correspond to 30-40 years in humans, or equivalent to a patient with hypertension for 15-20 years. Compared to the negative control group, the PEG conjugates of kinin and the valsartan groups showed significant or extremely significant decreases in blood pressure. See the table below for details.

[0134] Table 8-2 Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0135] Compared with the negative control group, the Lys-BKm13-SPA10K group showed a significant (P<0.05) or extremely significant (P<0.01) decrease in the aortic media collagen volume fraction (CVF), while the aortic media elastic fiber volume fraction (EVF) showed an extremely significant (P<0.01) increase. This indicates that Lys-BKm13-SPA10K administration can significantly improve the pathological changes in aortic tissue caused by hypertension. See the table below, Figures 7 and 8 for details:

[0136] Table 8-3 Changes in CVF and EVF of animals in each group (Mean±SD, %) Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0137] Example 9 Comparison of the blood pressure lowering effect of long-acting BK in spontaneously hypertensive rats (4)

[0138] In the present invention, spontaneously hypertensive rats (SHR) were used as model animals to compare the blood pressure-lowering effects of a series of drugs in SHR rats via different administration routes (intramuscular injection, subcutaneous injection, and oral administration).

[0139] 1. Grouping and Experimental Design

[0140] SHR rats were used as model animals, with a total of 140 male SHR rats and 20 male WKY rats (as normal blood pressure control group), 13-15 weeks old. There were 8 groups in the experiment, namely model group, valsartan group, Lys-BKm13-SPA5K low-dose group, Lys-BKm13-SPA5K medium-dose group, Lys-BKm13-SPA5K high-dose group, Lys-BKm13-SPA10K subcutaneous group, and WKY negative control group, with 20 animals in each group. The oral administration group was administered 5 times a week, the subcutaneous administration group was administered 2 times a week, and the intramuscular administration group was administered once a week for a total of 8 weeks. The systolic blood pressure, diastolic blood pressure and mean arterial pressure of all animals were tested the next day after completing 4 or 8 weeks of administration. The grouping and administration information are shown in the following table:

[0141] Table 9-1 Note: a: number of animals.

[0142] 2. Results

[0143] SHR rats enter the hypertension establishment stage after 16 weeks of age, and their blood pressure reaches the highest level at 24 weeks of age. 13 to 23 weeks of age corresponds to 16 to 25 years old in humans or is equivalent to patients with initial hypertension. The systolic blood pressure, mean arterial pressure, and diastolic blood pressure of each group of SHR rats were significantly (P<0.01) higher than those of the WKY group. After 4 or 8 weeks of continuous administration, the systolic blood pressure, mean arterial pressure, and diastolic blood pressure of animals in the oral Lys-BKm13-SPA5K or subcutaneous Lys-BKm13-SPA10K administration groups decreased significantly (P<0.05) or significantly (P<0.01) compared with the model group animals, or there was a certain downward trend; there was a certain dose-effect relationship between the low, medium, and high dose groups of Lys-BKm13-SPA5K. After 4 weeks of administration or 8 weeks of administration, the blood pressure changes of animals in each group are detailed in the table below.

[0144] Table 9-2 Changes in blood pressure of animals in each group after 4 weeks of drug administration (Mean ± SEM, mmHg) Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0145] Table 9-3 Changes in blood pressure of animals in each group after 8 weeks of drug administration (Mean ± SEM, mmHg) Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0146] Example 10 Comparison of the blood pressure lowering effect of long-acting BK in spontaneously hypertensive rats (5)

[0147] In the present invention, spontaneously hypertensive rats (SHR) were used as model animals to compare the blood pressure-lowering effects of a series of drugs administered via different routes (subcutaneous and oral).

[0148] 1. Grouping and Experimental Design

[0149] SHR rats were used as model animals, with a total of 140 male SHR rats and 20 male WKY rats, aged 23-25 ​​weeks. There were 8 groups in the experiment, namely the model group, valsartan group, Lys-BKm13-SPA5K low-dose group, Lys-BKm13-SPA5K medium-dose group, Lys-BKm13-SPA5K high-dose group, Lys-BKm13-SPA10K subcutaneous low-dose group, Lys-BKm13-SPA10K subcutaneous high-dose group, and WKY negative control group, with 20 animals in each group. The oral administration group was given 5 times a week, and the subcutaneous administration group was given 2 times a week for a total of 8 weeks. The systolic blood pressure, diastolic blood pressure and mean arterial pressure of all animals were tested the next day after completing 4 or 8 weeks of administration; the serum of rats after the last dose was collected to detect the levels of nitric oxide and creatinine in the serum. The grouping and administration information are shown in the following table: Note: a: number of animals.

[0150] 2. Results

[0151] Hypertension is established in SHR rats after 16 weeks of age, with blood pressure reaching its highest level at 24 weeks of age. Ages 23 to 33 weeks correspond to 25 to 30 years in humans, or equivalent to a patient with hypertension for 8 to 12 years. (For the correlation between rat age and human age, see Points to Consider in Designing and Conducting Juvenile Toxicology Studies.)

[0152] International Journal of Toxicology 2017, Vol. 36(4) 325-339) The systolic blood pressure, mean arterial pressure, and diastolic blood pressure of SHR rats in each group were significantly higher (P<0.01) than those in the WKY group. After 4 or 8 weeks of continuous administration, the systolic blood pressure, mean arterial pressure, and diastolic blood pressure of animals in the oral Lys-BKm13-SPA5K or subcutaneous Lys-BKm13-SPA10K groups decreased significantly (P<0.05) or significantly (P<0.01) compared with the model group, or there was a certain downward trend; there was a certain dose-effect relationship between the low, medium, and high doses of Lys-BKm13-SPA5K. After 4 or 8 weeks of administration, the changes in blood pressure of animals in each group are shown in the table below.

[0153] Table 10-1 Changes in blood pressure of animals in each group after 4 weeks of drug administration (Mean ± SEM, mmHg) Note: **: P < 0.01, vs. model group; *: P < 0.05, vs. model group.

[0154] Table 10-2 Changes in blood pressure of animals in each group after 8 weeks of drug administration (Mean ± SEM, mmHg) Note: **: P < 0.01, vs. negative control group; *: P < 0.05, vs. negative control group.

[0155] Examples 6-10 all adopted the SHR model, which is a relatively recognized experimental animal model in the study of hypertension and its complications; the test drugs used showed good activity (receptor binding activity and signal transduction activity) in the in vitro cell activity experiment (Example 5), and after administration to SHR model rats of different ages, they could lower blood pressure and improve complications such as vascular lesions caused by hypertension.

[0156] In addition to the test drugs in Examples 6-8, when other kinin derivatives that showed good activity (receptor binding activity and signal transduction activity) in in vitro cell activity experiments were administered to SHR model rats (such as Lys-BKm01, m02, m06, m12, and m14 using other suitable PEG conjugates), similar effects to those in Examples 6-8 were observed, namely, lowering the blood pressure of SHR rats and improving complications such as vascular lesions caused by hypertension; on the contrary, when kinin derivatives that lost more of the above-mentioned receptor binding activity and signal transduction activity were administered (such as PEG conjugates of Lys-BKm05, m07, etc.), no similar effects were observed.

[0157] In summary, kinin derivatives that better retain receptor binding activity and signal transduction activity can be used to treat hypertension in patients of different ages and years of illness, and to improve complications such as vascular lesions caused by hypertension.

[0158] It should be noted that polypeptide drugs generally have the problems of poor stability and short half-life in vivo, and lysyl bradykinin and bradykinin are more unstable than ordinary polypeptides, with a half-life of only a few seconds. Exogenously administered bradykinin is quickly hydrolyzed and inactivated by kininase in the body and cannot be made into a drug; at the same time, large doses of exogenous bradykinin / lysyl bradykinin entering the body will cause a rapid drop in blood pressure and adverse reactions such as hypotension. For the above reasons, bradykinin / lysyl bradykinin is not druggable. The long-acting lysyl bradykinin and bradykinin of this application fundamentally solve the problem of bradykinin / lysyl bradykinin being druggable. Although the long-acting methods exemplified in the embodiments are all PEG coupling, the long-acting methods of lysyl bradykinin and bradykinin are not the primary problem to be solved by this application. The PEG coupling technology used in the specific embodiments is only used to exemplify and not to limit the long-acting methods of this application. Based on the disclosure of this application, those skilled in the art can anticipate that the use of other long-acting technologies while better retaining receptor binding activity and signal transduction activity of lysyl bradykinin / bradykinin derivatives can reduce SHR blood pressure and improve complications such as vascular lesions caused by hypertension.

[0159] In addition, the embodiment cites the stability, in vitro activity, and in vivo drug efficacy of the PEG modifications of lysyl bradykinin or bradykinin and its various variants. The results all show good stability, and the lysyl bradykinin or bradykinin variants that retain in vitro activity can all solve the problem of extending the half-life of lysyl bradykinin or bradykinin and exerting its in vivo biological activity after being modified with polyethylene glycol; On the contrary, other lysyl bradykinins or bradykinin variants that do not retain in vitro activity are modified with polyethylene glycol, or lysyl bradykinin or bradykinin and its various variants (without PEG modification) have no drug efficacy in vivo. Therefore, those skilled in the art can reasonably expect that other long-acting lysyl bradykinins or bradykinin derivatives that retain receptor binding activity can all solve the problem of extending the half-life of lysyl bradykinin or bradykinin and exert its in vivo biological activity, reduce SHR model animal blood pressure, improve hypertensive complications, and are used to treat hypertension, prevent or treat complications such as vascular lesions caused by hypertension. It is not limited to the specific lysyl bradykinin or bradykinin variants cited in the embodiment.

Claims

1. Use of kinin or its derivatives in the preparation of a drug for treating hypertension or preventing or treating complications of hypertension, wherein the kinin is bradykinin or lysyl bradykinin.

2. Use of the kinin or its derivative according to claim 1 in the preparation of a drug for treating hypertension or preventing or treating complications of hypertension, wherein the kinin is a long-acting kinin, and the kinin derivative is a long-acting kinin derivative.

3. Use of the kinin or its derivative according to claim 1 in the preparation of a drug for treating hypertension or preventing or treating complications of hypertension, wherein the kinin derivative is a polyethylene glycol-modified kinin, or a kinin mutant, or a fusion protein formed by fusion of kinin with an Fc fragment or with human serum albumin, or kinin conjugated with a fatty acid, or a long-acting preparation of kinin.

4. Use of the kinin or its derivatives according to claim 3 in the preparation of a drug for treating hypertension or preventing or treating complications of hypertension, wherein the PEG modifier used in the polyethylene glycol modification of the kinin is a SPA, SCM or SS modifier, and the PEG modifier is a linear or branched PEG modifier.

5. Use of the kinin or its derivatives according to claim 4 in the preparation of a drug for treating hypertension or preventing or treating complications of hypertension, wherein the molecular weight of the PEG modifier is 2KD-20KD.

6. Use of the kinin or its derivatives according to claim 3 in the preparation of a drug for treating hypertension, or preventing or treating complications of hypertension, wherein the mutant of the kinin is a cysteine ​​introduced at any position in the lysyl bradykinin or bradykinin sequence, and / or an arbitrary amino acid is inserted between the first and second amino acid residues at the N-terminus of the lysyl bradykinin, and / or an arbitrary amino acid is inserted before the first amino acid residue at the N-terminus of the bradykinin; and / or it is composed of 2 or more lysyl bradykinin monomers connected in series, or 2 or more bradykinin monomers connected in series, or a lysyl bradykinin monomer and a bradykinin monomer connected in series; and / or Phe at position 6 of lysyl bradykinin or Phe at position 5 of bradykinin is mutated to other amino acids.

7. A pharmaceutical composition for treating hypertension, or preventing or treating complications of hypertension, comprising kinin or its derivatives, wherein the kinin is bradykinin or lysyl bradykinin.

8. The pharmaceutical composition for treating hypertension or preventing or treating complications of hypertension according to claim 7, wherein the kinin is a long-acting kinin, and the kinin derivative is a long-acting kinin derivative.

9. The pharmaceutical composition for treating hypertension or preventing or treating complications of hypertension according to claim 7, wherein the kinin derivative is a polyethylene glycol-modified kinin, or a kinin mutant, or a fusion protein formed by fusion of kinin with an Fc fragment or with human serum albumin, or kinin conjugated with a fatty acid, or a long-acting preparation of kinin.

10. The pharmaceutical composition for treating hypertension, or preventing or treating complications of hypertension according to claim 9, wherein the PEG modifier used in the polyethylene glycol modification of the kinin is a SPA, SCM or SS modifier, and the PEG modifier is a linear or branched PEG modifier.

11. The pharmaceutical composition for treating hypertension, or preventing or treating complications of hypertension according to claim 10, wherein the molecular weight of the PEG modifier is 2KD-20KD.

12. The pharmaceutical composition for treating hypertension, or preventing or treating complications of hypertension according to claim 9, wherein the mutant of the bradykinin is a mutant in which cysteine ​​is introduced at any position in the lysyl bradykinin or bradykinin sequence, and / or any amino acid is inserted between the first and second amino acid residues at the N-terminus of the lysyl bradykinin, and / or any amino acid is inserted before the first amino acid residue at the N-terminus of the bradykinin; and / or it is composed of 2 or more lysyl bradykinin monomers connected in series, or 2 or more bradykinin monomers connected in series, or lysyl bradykinin monomers and bradykinin monomers connected in series; and / or Phe at position 6 of lysyl bradykinin or Phe at position 5 of bradykinin is mutated to other amino acids.

13. A method for treating hypertension, or preventing or treating complications of hypertension, comprising administering kinin or its derivatives to a patient, wherein the kinin is bradykinin or lysyl bradykinin.

14. The method for treating hypertension, or preventing or treating complications of hypertension according to claim 13, wherein the kinin is a long-acting kinin, and the kinin derivative is a long-acting kinin derivative.

15. The method for treating hypertension, or preventing or treating complications of hypertension according to claim 13, wherein the kinin derivative is a polyethylene glycol-modified kinin, or a kinin mutant, or a fusion protein formed by fusion of kinin with an Fc fragment or with human serum albumin, or kinin conjugated with a fatty acid, or a long-acting preparation of kinin.

16. The method for treating hypertension, or preventing or treating complications of hypertension according to claim 15, wherein the PEG modifier used in the polyethylene glycol modification of the kinin is a SPA, SCM or SS modifier, and the PEG modifier is a linear or branched PEG modifier.

17. The method for treating hypertension, or preventing or treating complications of hypertension according to claim 16, wherein the molecular weight of the PEG modifier is 2KD-20KD.

18. The method for treating hypertension, or preventing or treating complications of hypertension according to claim 15, wherein the mutant of the bradykinin is a mutant in which cysteine ​​is introduced at any position in the lysyl bradykinin or bradykinin sequence, and / or any amino acid is inserted between the first and second amino acid residues at the N-terminus of the lysyl bradykinin, and / or any amino acid is inserted before the first amino acid residue at the N-terminus of the bradykinin; and / or it is composed of 2 or more lysyl bradykinin monomers connected in series, or 2 or more bradykinin monomers connected in series, or lysyl bradykinin monomers and bradykinin monomers connected in series; and / or Phe at position 6 of lysyl bradykinin or Phe at position 5 of bradykinin is mutated to other amino acids.

Images