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

Abstract

The 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 of the long-acting kinin 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

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 the leading cause of premature death and cardiovascular disease worldwide. The prevalence of hypertension among adults in my country is about 27.5%. In recent years, the awareness, treatment and control rates of hypertension patients in my country have achieved good results, reaching 51.6%, 45.8% and 16.8% respectively, but are still at a relatively low level overall.

[0003] Hypertension refers to a clinical syndrome characterized by increased systemic arterial blood pressure (systolic and / or diastolic blood pressure) (the 2022 "Chinese Hypertension Clinical Practice Guidelines" 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 deficiencies in lowering blood pressure and improving symptoms. Modern medicine still has many unknowns about the exact cause and pathogenesis of hypertension, and it is very necessary to develop more effective therapeutic drugs. Summary of the invention

[0005] 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 preparing drugs for treating or improving hypertension.

[0006] The present invention also provides a pharmaceutical composition for treating or improving hypertension, comprising kinin or its derivatives.

[0007] The present invention also provides a method for treating or improving hypertension, which comprises administering kinin or its derivatives to a patient.

[0008] 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 drugs for preventing or treating complications of hypertension.

[0009] The present application also provides a pharmaceutical composition for preventing or treating complications of hypertension, comprising kinin or its derivatives.

[0010] The present invention also provides a method for preventing or treating complications of hypertension, which comprises administering kinin or its derivatives to a patient.

[0011] The complications of hypertension include hypertensive heart and vascular disease, hypertensive encephalopathy, hypertensive nephropathy, etc.

[0012] The above-mentioned kinin includes bradykinin or lysyl bradykinin.

[0013] The kinin or its derivative is a long-acting kinin or its derivative.

[0014] 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.

[0015] The administration method includes, but is not limited to, injection administration, oral administration, etc. The injection administration method includes, but is not limited to, intravenous injection, subcutaneous injection, etc.

[0016] Peptide drugs generally have the problems of poor stability and short half-life in vivo, especially drugs for the treatment of chronic diseases often require long-term and frequent administration. The degradation of lysyl bradykinin / bradykinin in the body is mainly participated by aminopeptidases, carboxypeptidases and angiotensin ACEII, and the dynamic balance of bradykinin in the body is achieved based on the kallikrein and degradation enzyme system. Compared with ordinary peptides, lysyl bradykinin and bradykinin are more unstable. It has been reported that their half-life is only a few seconds. Exogenously administered bradykinin is quickly hydrolyzed and inactivated by kininase in the body.

[0017] 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 of fatty acids; 2. Making peptide drugs long-acting through formulation means.

[0018] In the specific examples of the present 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.

[0019] Regarding polyethylene glycol modification, in the specific embodiments of the present application, the modifiers used include SPA, SCM, and SS modifiers; they can be straight-chain or branched PEG modifiers.

[0020] Preferably, the PEG modifier is a linear PEG modifier.

[0021] Preferably, the molecular weight of the PEG modifier is 2KD-20KD.

[0022] Preferably, the molecular weight of the PEG modifier is 5KD-10KD.

[0023] In the optimization of the amino acid sequence of bradykinin, 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.

[0024] In a specific embodiment of the present application, any number and any type of amino acids are inserted between the first and second amino acid residues at the N-terminus of wild-type lysyl bradykinin, i.e., KXRPPGFSPFR, 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 wild-type bradykinin, i.e., XRPPGFSPFR, where X represents any number and any type of amino acids. The 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.

[0025] In a specific embodiment of the present application, a lysyl bradykinin or bradykinin derivative is formed by connecting two or more lysyl bradykinin monomers in series (such as m14), or by connecting two or more bradykinin monomers in series, or by connecting a lysyl bradykinin monomer and a bradykinin monomer 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.

[0026] In a specific embodiment of the present application, the wild-type lysyl bradykinin Phe at position 6 or the wild-type bradykinin Phe at position 5 is mutated to other amino acids, such as unnatural amino acids Igl (α-2-indolylglycine) and THI (β-2-thiophenylalanine). The LBK or BK after the above site mutation is not easily recognized by the enzyme in the body, thereby effectively extending its half-life in the body.

[0027] Preferably, the lysyl bradykinin or bradykinin derivative of the present application has a sequence as shown in SEQ ID NO:14.

[0028] The polyethylene glycol modification method and amino acid sequence optimization used in the specific embodiments of the present application are only used to illustrate that the half-life of kinin can be extended by the above method, but 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 the present application, or use other polypeptide drug long-acting modification methods (such as fusion with Fc fragment, fusion with human serum albumin, conjugation with fatty acids, preparations as described above) to modify kinin to make it long-acting.

[0029] Spontaneously hypertensive rat (SHR) is the preferred animal model for screening antihypertensive drugs. The systolic blood pressure of normal rats is 110-120 mmHg, while the peak blood pressure of SHR can reach 180-200 mmHg after breeding.

[0030] After birth, the blood pressure of SHR increases with the age of the mouse. As the disease progresses, SHR may develop complications such as heart, brain, and kidney. Therefore, on the basis of its application in the screening of hypertension drugs, the SHR model is also increasingly used in the study of other diseases.

[0031] Left ventricular hypertrophy and myocardial fibrosis are common complications of hypertension and are also independent risk factors for arrhythmias, heart failure and sudden death. Early control of blood pressure, prevention and reversal of left ventricular hypertrophy have become the focus of prevention and treatment of hypertension. SHR has hypertensive heart and vascular lesions. Several studies have confirmed that SHR is an ideal animal model for studying hypertensive left ventricular hypertrophy and can be used to study cardiac dysfunction caused by hypertension.

[0032] SHR is also a model of hypertensive brain damage. The time-dependent increase in SHR arterial blood pressure, brain atrophy, and neuronal cell loss are similar to the occurrence of human hypertensive encephalopathy to a certain extent.

[0033] In addition, long-term hypertension can lead to renal arteriosclerosis, proteinuria, renal dysfunction, and severe renal failure. Several studies have confirmed that SHR is an ideal model for studying hypertensive nephropathy.

[0034] The results of the present application show that kinin or its derivatives can significantly reduce blood pressure and significantly improve the heart, brain and kidney functions of SHR after administration. Therefore, kinin or its derivatives can be used to treat hypertension and prevent complications of hypertension.

[0035] The kallikrein-kinin system, referred to as KKS, is involved in a variety of physiological and pathological processes, such as the regulation of cardiovascular, renal and nervous system functions. KKS includes kallikrein (KLK), kininogen, kinin, kinin receptors (B1, B2 receptors) and kininase. Kallikrein, also known as kininogenase or kallikrein, is a serine protease, which is divided into two major categories: plasma kininogenase (PK) and tissue kininogenase (TK), both of which play very important physiological roles. It is currently believed that human tissue kininogenase consists of at least 15 members (KLK1-KLK15), among which tissue kininogenase 1 (KLK1) has been studied more. KLK1 converts kininogen into kinin and acts on the corresponding receptors to play a series of biological effects. The kinins that have been studied more clearly at present are lysyl bradykinin (10 peptides) and bradykinin (9 peptides). The former is cleaved into bradykinin by aminopeptidase in the body. Both have multiple physiological functions such as regulating blood pressure and inflammatory response. 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, a large dose 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. This application study found that long-acting kinin or its derivatives can fundamentally solve the problem of bradykinin / lysyl bradykinin being drug-free. 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 and hypotension in a short period of time. Adverse reactions. The long-acting kinin or its derivatives of this application are used to treat hypertension and prevent hypertension complications, providing a new method for treating hypertension and preventing hypertension complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :Lys-BK and its variants activate the phosphorylation of downstream ERK1 / 2 and CREB proteins, among which β-actin is the internal reference; p- represents the phosphorylation of the corresponding protein; control is the buffer blank control; Lys-BK-M06-EH is the sample after m06 mixed with serum

[0037] Figure 2 :Detection of the binding activity of Lys-BK and its variant PEG conjugates to B2 receptor (reporter gene method)

[0038] Figure 3:Lys-BK and its variant PEG conjugates activated the phosphorylation of downstream ERK1 / 2 and CREB proteins, among which β-actin is the internal reference; p- represents the phosphorylation of the corresponding protein; control is the buffer blank control

[0039] Figure 4 Example 6 Effect of the Test Drug on Blood Pressure in Spontaneously Hypertensive Rats

[0040] Figure 5 Example 7 Effect of the Test Drug on Blood Pressure in Spontaneously Hypertensive Rats

[0041] Figure 6 Example 8 Effect of the Test Drug on Blood Pressure in Spontaneously Hypertensive Rats DETAILED DESCRIPTION

[0042] Unless otherwise specified, the technical terms or abbreviations in this application have the following meanings:

[0043] Lys-BK: Wild-type lysyl bradykinin, which is consistent with the sequence of natural human lysyl bradykinin; the amino acid sequence is KRPPGFSPFR.

[0044] BK: wild-type bradykinin, which is consistent with the sequence of natural human bradykinin; the amino acid sequence is RPPGFSPFR.

[0045] Mutant: A variant obtained by replacing, inserting or deleting some amino acids based on wild-type lysyl-bradykinin or bradykinin.

[0046] Derivatives: include not only the various mutants listed 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 obtained by further modification based on the Lys-BK / BK mutants of the present application.

[0047] Polyethylene glycol: PEG, usually polymerized by ethylene oxide, has branched, linear and multi-arm types. 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.

[0048] Polyethylene glycol modifier: PEG modifier refers to a polyethylene glycol derivative with a functional group. It is an activated polyethylene glycol that can be used for protein and peptide drug modification. The polyethylene glycol modifier used in this application is 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 labeled value, such as the molecular weight of PEG5K can be 4.5kDa to 5.5kDa.

[0049] M-SPA-5K: a linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 5 kDa, the structure of which is shown in formula (1), wherein n is an integer from 98 to 120.

[0050]

[0051] M-SPA-2K: a linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 2 kDa, a structure as shown in formula (1), and n is an integer from 37 to 45.

[0052] M-SPA-10K: a linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 10 kDa, a structure as shown in formula (1), and n is an integer from 200 to 245.

[0053] M-SPA-20K: a linear monomethoxy polyethylene glycol succinimidyl propionate with a molecular weight of about 20 kDa, a structure as shown in formula (1), and n is an integer from 405 to 495.

[0054] 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), and n is an integer from 99 to 120.

[0055]

[0056] M-SS-5K: a linear monomethoxy polyethylene glycol succinimidyl succinate with a molecular weight of about 5 kDa, a structure as shown in formula (3), and n is an integer from 98 to 119.

[0057]

[0058] 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,

[0059]

[0060] Example 1 Design and Synthesis of Lysyl-Bradykinin / Bradykinin Variants

[0061] Based on the sequence of lysyl bradykinin / bradykinin and possible degradation pathways in vivo, a series of variants were designed (Table 1-1), mainly replacing with natural amino acids and non-natural amino acids with similar structures (m01-m04, m07, where m02 is a variant of BK); in order to confirm the minimum active unit for biological activity / function, partially truncated or extended polypeptide variants (m05, m06, m08-m11) were designed; in order to further improve the antioxidant properties of the variants, cysteine ​​(m13, m15, m16) was introduced; in order to further improve the stability of the polypeptide, lysyl bradykinin / bradykinin monomers were connected in series (m12 is Lys-BK connected in series with BK, and m14 is two Lys-BK connected in series); based on the m13 polypeptide, the effect of N-terminal extension on drug efficacy was compared, and m15 and m16 variants were designed. The purity of all polypeptides is >95%.

[0062] Table 1-1 Lysyl bradykinin / bradykinin variant sequences

[0063]

[0064]

[0065] Example 2 Study on the stability of lysyl bradykinin / bradykinin and its variants

[0066] 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, incubated at 37°C for 30 min, 60 min, 90 min, 120 min, 180 min and other different time points, and terminated by adding 3% trichloroacetic acid (50% volume of the above peptide serum mixed system) to effectively remove the matrix interference in the serum. Further based on UPLC-RP-UV 214nm Methods The degradation of peptide samples in serum system was monitored to evaluate their stability. 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.

[0067] Table 2-1 Stability of lysyl bradykinin and its variants in serum system

[0068] Peptide name Serum system residual time Lys-BK 30~60min Lys-BKm01 60~90min Lys-BKm02 60~90min Lys-BKm03 60~90min Lys-BKm04 >180min Lys-BKm05 >180min Lys-BKm06 60~90min Lys-BKm07 60~90min Lys-BKm11 60~90min Lys-BKm12 90-120min Lys-BKm13 60~90min Lys-BKm14 90-120min Lys-BKm15 60~90min Lys-BKm16 60~90min

[0069] The results in Table 2-1 show that the designed polypeptide variants are more stable in serum than wild-type lysyl bradykinin, which also indicates that they have certain advantages in stability and efficacy in vivo.

[0070] Example 3 Evaluation of in vitro activity of lysyl bradykinin / bradykinin and its variants

[0071] 1. B2 receptor binding activity based on reporter gene method

[0072] The in vivo pharmacological effects of lysyl-bradykinin / bradykinin are achieved by binding to the B2 receptor and activating the downstream signaling pathway. A B2 receptor reporter gene stably transfected cell line B2-NFAT-CHO-K1 was constructed based on CHO-K1 host cells and the cell library was completed, which was used for the in vitro detection and evaluation of the binding activity of lysyl-bradykinin / bradykinin or its variants, as well as PEG modifications, to the B2 receptor.

[0073] Detection method: cells were plated at 5×10 4 The density of 1000 μg / well was spread on a 96-well plate, and the samples were cultured at 37°C and 5% CO2 for 16-20 hours. Then, the standard and test solutions were diluted in gradients. After 5 hours, the Bio-Lite luciferase detection reagent was added to measure the chemiluminescence intensity. GraphPad was used to draw a graph, and the four-parameter equation curve was fitted according to EC. 50 The relative activity was calculated based on the values. Table 3-1 of some test results lists the relative binding activity of the peptide variants to the B2 receptor. Among them, the activity of Lys-BKm11, Lys-BKm13, Lys-BKm15 and Lys-BKm16 variants was significantly improved compared with the wild-type lysyl bradykinin.

[0074] Table 3-1 Relative binding activity of Lys-BK and its variants to B2 receptor

[0075] Peptide name Relative activity Peptide name Relative activity Lys-BK 100％ Lys-BKm08 <10％ Lys-BKm01 100％ Lys-BKm09 0％ Lys-BKm02 100％ Lys-BKm10 0％ Lys-BKm03 <10％ Lys-BKm11 110％ Lys-BKm04 <10％ Lys-BKm12 100％ Lys-BKm05 <10％ Lys-BKm13 115％ Lys-BKm06 90％ Lys-BKm14 100％ Lys-BKm07 <10％ Lys-BKm15 125％ Lys-BKm16 120％

[0076] 2. Activity analysis of key target proteins in downstream signaling pathways

[0077] Based on B2-NFAT-CHO-K1 cells overexpressing B2 receptors, Lys-BK / BK or its different variants were given in vitro for intervention, and the changes of B2 receptor downstream signals p-CREB and p-ERK1 / 2 were detected by WB method (P represents phosphorylation, serine or threonine on the protein is activated after phosphorylation, and a series of effects are produced in the pathway) to compare the activity differences of the test substances. Extracellular signal-regulated kinases (ERK1 / 2) and cAMP-response element binding protein (CREB) are both signal molecules that directly act downstream after B2R activation. After stroke, the activation of CREB and ERK1 / 2 can prevent the occurrence of post-ischemic inflammatory response and neuronal damage. Therefore, the level of their expression can reflect the ability of the test substance to activate B2R to a certain extent, and at the same time reflect the significance of neuroprotective effect from the side.

[0078] CHO-K1 (B2R+) cells in the logarithmic growth phase were collected and adjusted to 3×10 6 cells / mL, inoculated in cell culture plates, 3×10 5 / well. Incubate in a CO2 incubator at 37.0℃ and 5.0% CO2 for 24 to 48 hours. When the cell density grows to more than 90%, wash with PBS, add 1ml 1μM Lys-BK / BK or its variant sample to each well, and the control group is F12 medium. Place the cell culture plate in a 37.0℃, 5.0% CO2 incubator for 5 minutes, and wash the cell culture plate with PBS. Add 80μL / well of protein lysis buffer (RIPA, PMSF, phosphatase inhibitors), and grind the bottom of the well back and forth with a scraper to accelerate cell lysis. Transfer the cell fragments and lysis buffer to a centrifuge tube, centrifuge at 12000rpm, 10min, 4℃. Aspirate the supernatant, mix with 5× Loading Buffer at a volume ratio of 4:1, and boil in a metal bath at 100℃ for 10min. Load an equal amount of protein sample onto 10% SDS-PAGE for gel running, membrane transfer, blocking, antibody incubation, luminescence identification and other operations.

[0079] The results of the detection of downstream signaling activity activated by Lys-BK / BK or its variants are as follows Figure 1As shown, lysyl bradykinin 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 sample is qualitatively evaluated by judging the phosphorylation level of downstream ERK1 / 2 and CREB proteins. The evaluation results show that Lys-BKm05 and Lys-BKm07 variants have lost the activity of activating downstream pathways, and LBKm01, m02, m06, m11, m12, m13, m14, m15, and m16 all show the activity of activating downstream pathways.

[0080] Based on the above two in vitro activity evaluation results, it can be known that: after the C-terminal truncation of lysyl bradykinin / bradykinin, the binding activity and signal transduction activity with B2R are lost, the lack of N-terminal lysine of lysyl bradykinin does not affect its binding activity and signal transduction activity with B2R, and the loss of other amino acids at the N-terminal loses the binding activity and signal transduction activity with B2R. The minimum activity unit is RPPGFSPFR; N-terminal extension (m11) or introduction of cysteine ​​(m13) are beneficial to improve the binding activity with B2R. Figure 1 d It can be seen that the phosphorylation of CREB protein in the m12, 13, and 14 groups was significantly higher than that in the LBK group, revealing that m12, 13, and 14 can better activate downstream pathways and show in vitro activity superior to LBK.

[0081] Example 4 Preparation of PEG conjugates of lysyl bradykinin / bradykinin and its variants

[0082] The PEG modification of lysyl bradykinin / bradykinin and its variants is carried out by conventional methods in the art. For specific variants and specific PEG modifiers, some parameters have slight changes, which can be obtained by those skilled in the art through limited experimental optimization, and there are no technical obstacles. Taking M-SPA-5K modified wild-type BK as an example, the preparation process is as follows:

[0083] 1. Preparation of PEG conjugate samples

[0084] The peptide sample was dissolved in sodium dihydrogen phosphate / sodium hydrogen phosphate buffer pH 7.0 to a final concentration of 10 mg / mL. PEG was added to the peptide solution at a mass ratio of 1:7 between the peptide and M-SPA-5K, and the mixture was stirred slowly until uniformly mixed, and then reacted at 2-8°C for 1 h.

[0085] 2. Purification of reaction mixture

[0086] The first step of purification chromatography conditions are as follows:

[0087] 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.

[0088] Sample loading: directly load the above polypeptide-PEG modification reaction mixture after the reaction is completed.

[0089] Elution: After loading, wash the column with mobile phase for at least 1.5 column volumes. 214nm Trend Collect elution samples in steps.

[0090] The second step purification chromatography conditions are as follows:

[0091] 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.

[0092] 3. Purity analysis of PEG conjugate samples

[0093] (1) HPLC purity analysis

[0094] The detection was carried out with reference to the general rule 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.

[0095] 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.

[0096] 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%.

[0097] (2) SDS-PAGE purity analysis

[0098] 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 configured for sample testing.

[0099] The electrophoresis results showed that the prepared lysyl bradykinin variant series PEG-modified products had uniform bands without any mixed bands, and the purity was >95%.

[0100] 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.

[0101] Example 5 In vitro evaluation of PEG conjugates of lysyl bradykinin / bradykinin and its variants

[0102] 1. Stability

[0103] The stability of the PEG conjugates of lysyl bradykinin and its variants was indirectly evaluated by evaluating the stability in the in vitro serum system, and the detection method was as in Example 2. The residual time of the PEG conjugates of lysyl bradykinin and its variants in the 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.

[0104] Table 5-1 Stability of PEG conjugates of lysyl bradykinin and its variants in serum system

[0105] Peptide name Serum system residual time Lys-BK-SPA5K >6h Lys-BKm01-SPA2K >6h Lys-BKm01-SPA5K >6h Lys-BKm01-SPA10K >6h Lys-BKm01-YNHS10K >12h Lys-BKm01-SCM5K >6h Lys-BKm01-SS5K >6h

[0106] 2. In vitro activity

[0107] The PEG conjugates of lysyl bradykinin and its variants were tested for in vitro B2 receptor binding activity and signal transduction activity, and the detection method was the same as in Example 3. Lysyl bradykinin or its variants still have B2 receptor binding activity after PEG modification ( Figure 2 a, 2b), while still activating the phosphorylation of key proteins in the downstream signaling pathway of the B2 receptor ( Figure 3 ). The coupling products of PEG modifiers of different structural types and different molecular weights all retain good binding activity with B2 receptors. The PEG modifiers include: molecular weights ranging from 2K to 20K, straight chain and branched, and different structures (SPA, SCM, SS, etc.). The results show that the activity of straight chain PEG conjugates is higher than that of branched chain PEG conjugates at the same molecular weight, and this result is true for both 10K and 20K molecular weight PEG. Figure 2 As shown in a, the EC50 values ​​of Lys-BKm01-SPA10K and Lys-BKm01-YNHS10K were 90.8μg / ml and 346.6μg / ml, respectively, and the activity of the linear PEG conjugate was nearly 4 times higher than that of the branched PEG conjugate. In addition, under the same PEG structure type, the PEG conjugates of 2K and 10K were relatively more active among the four PEGs of 2K, 5K, 10K, and 20K.

[0108] Results of PEG conjugates of lysyl bradykinin and its variants activating downstream signaling pathways ( Figure 3) showed that the peptide still retained the activity of activating ERK1 / 2 and CREB phosphorylation downstream of B2 receptor after PEG conjugation, although it was lower than that of Lys-BK peptide, especially the phosphorylation of ERK1 / 2. Under the same SPA5K-PEG modifier, the activity of Lys-BKm13-SPA5K among different peptide variants was relatively the highest; under the same peptide variant Lys-BKm01, the activity of 2K and 10K PEG-modified conjugates was relatively 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.

[0109] In summary, the stability of PEG conjugates of lysyl bradykinin and its variants is significantly improved, and the B2 receptor binding activity and signal transduction activity before PEG conjugation are still well retained.

[0110] Example 6 Comparison of the hypotensive effects of PEG conjugates of kinin in spontaneously hypertensive rats (1)

[0111] 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 different administration routes (subcutaneous, oral) on SHR rats.

[0112] 1. Grouping and Experimental Design

[0113] SHR rats were used as model animals, with a total of 100 male SHR rats, 16-18 weeks old. There were 4 groups in the experiment, namely the negative control group, the Lys-BKm13-SPA10K group (the test drug was a PEG conjugate of kinin, the kinin was Lys-BKm13, modified with M-SPA-10K), the Lys-BKm13-SPA5K subcutaneous group (the test drug was a PEG conjugate of kinin, the kinin was Lys-BKm13, modified with M-SPA-5K), and the 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 5 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 8 weeks of administration.

[0114] Table 6-1

[0115]

[0116]

[0117] Note: a: Number of animals.

[0118] 2. Results

[0119] SHR rats enter the hypertension establishment period after 16 weeks of age, and blood pressure reaches the highest level at 24 weeks of age. 16 to 24 weeks of age corresponds to 18 to 25 years old in humans or is equivalent to patients with initial hypertension. Compared with the negative control group, the blood pressure of the PEG conjugate group of kinin and the valsartan group decreased significantly or extremely significantly. See the table below for details:

[0120] Table 6-2

[0121]

[0122] Note: **: P < 0.01, vs negative control group; *: P < 0.05, vs negative control group.

[0123] Example 7 Comparison of the hypotensive effects of PEG conjugates of kinin in spontaneously hypertensive rats (2)

[0124] 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.

[0125] 1. Grouping and Experimental Design

[0126] 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 the 4-week drug administration. The grouping and dosing information are as follows:

[0127] Table 7-1

[0128]

[0129]

[0130] Note: a: Number of animals.

[0131] 2. Results

[0132] SHR rats enter the hypertension establishment period after 16 weeks of age, and blood pressure reaches the highest level at 24 weeks of age. 30 to 34 weeks of age corresponds to 25 to 30 years old in humans or equivalent to patients with hypertension for 8 to 12 years. Compared with the negative control group, the blood pressure of the PEG conjugate of kinin and valsartan group decreased significantly or extremely significantly. See the table below for details:

[0133] Table 7-2

[0134]

[0135] Note: **: P < 0.01, vs negative control group; *: P < 0.05, vs negative control group.

[0136] Example 8 Comparison of the hypotensive effects of PEG conjugates of kinin in spontaneously hypertensive rats (3)

[0137] 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.

[0138] 1. Grouping and Experimental Design

[0139] SHR rats were used as model animals, with a total of 100 male SHR rats, 40-42 weeks old. There were 4 groups in the experiment, namely the negative control group, the Lys-BKm13-SPA10K group, and the 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 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 the 4-week drug administration. The grouping and dosing information are as follows:

[0140] Table 8-1

[0141]

[0142] Note: a: Number of animals.

[0143] 2. Results

[0144] SHR rats enter the hypertension establishment period after 16 weeks of age, and blood pressure reaches the highest level at 24 weeks of age. 40 to 44 weeks of age corresponds to 30 to 40 years old in humans or equivalent to patients with hypertension for 15 to 20 years. Compared with the negative control group, the blood pressure of the Lys-BKm13-SPA10K group and the valsartan group decreased significantly or extremely significantly. See the table below for details:

[0145] Table 8-2

[0146]

[0147] Note: **: P < 0.01, vs negative control group; *: P < 0.05, vs negative control group.

[0148] Examples 6-8 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 being administered to SHR model rats of different ages, they could lower blood pressure and improve hypertension complications.

[0149] In addition to the test drugs of Examples 6-8, when other kinin derivatives (such as Lys-BKm01, m02, m06, m12, and m14 using other suitable PEG conjugates) that showed good activity (receptor binding activity and signal transduction activity) in in vitro cell activity experiments were administered to SHR model rats, similar effects to those of Examples 6-8 were observed, namely, lowering the blood pressure of SHR rats and improving hypertension complications; on the contrary, when kinin derivatives (such as PEG conjugates of Lys-BKm05, m07, etc.) that lost more of the above-mentioned receptor binding activity and signal transduction activity were administered, similar effects could not be observed.

[0150] In summary, kinin derivatives that better retain receptor binding activity and signal transduction activity can be used to treat hypertension and improve hypertension complications in patients of different ages and years of illness.

[0151] It should be noted that polypeptide drugs generally have 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 vivo and cannot be made into medicine; at the same time, large doses of exogenous bradykinin / lysyl bradykinin entering the body will cause blood pressure to drop rapidly, and adverse reactions such as hypotension will occur. 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 problems solved by this application. The PEG coupling technology used in the specific embodiments is only used to exemplify but not to limit the long-acting methods of this application. Based on the disclosure of this application, those skilled in the art can expect that the use of other long-acting technologies and lysyl bradykinin / bradykinin derivatives that better retain receptor binding activity and signal transduction activity can reduce SHR blood pressure and improve hypertension complications.

[0152] In addition, the embodiment exemplifies the stability, in vitro activity, and in vivo efficacy of the PEG modifiers of lysyl bradykinin or bradykinin and its various variants, and the results all show good stability, and the lysyl bradykinin or bradykinin variants with in vitro activity can be modified with polyethylene glycol to extend the half-life of lysyl bradykinin or bradykinin and exert its biological activity in vivo; on the contrary, other lysyl bradykinin or bradykinin variants without in vitro activity are modified with polyethylene glycol, or lysyl bradykinin or bradykinin and its various variants (without PEG modification) have no efficacy in vivo. Therefore, those skilled in the art can reasonably expect that other long-acting lysyl bradykinin or bradykinin derivatives with receptor binding activity can be solved to extend the half-life of lysyl bradykinin or bradykinin, exert its biological activity in vivo, reduce blood pressure of SHR model animals, improve hypertensive complications, and are used to treat hypertension, prevent or treat hypertensive complications. It is not limited to the specific lysyl bradykinin or bradykinin variants exemplified in the embodiment.

Claims

1. Use of kinin or its derivatives in the preparation of drugs for treating hypertension.

2. A pharmaceutical composition for treating hypertension, comprising kinin or its derivatives.

3. A method for treating hypertension, comprising administering kinin or its derivatives to a patient.

4. Use of kinin or its derivatives in the preparation of drugs for preventing or treating complications of hypertension.

5. A pharmaceutical composition for preventing or treating complications of hypertension, comprising kinin or its derivatives.

6. A method for preventing or treating complications of hypertension, comprising administering kinin or its derivatives to a patient.

7. The kinin according to any one of claims 1 to 6 is bradykinin or lysyl bradykinin.

8. The kinin derivative according to any one of claims 1 to 6, which is a long-acting kinin.

9. The kinin derivative according to claim 8 is a polyethylene glycol modified product of kinin, and the PEG modifier used is a SPA, SCM or SS modifier; the PEG modifier used is a linear or branched PEG modifier.

10. The kinin derivative according to claim 8 is a mutant of kinin, wherein 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 lysyl bradykinin, and / or any amino acid is inserted before the first amino acid residue at the N-terminus of 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.

11. The kinin derivative according to claim 8 is a fusion protein, wherein the kinin is fused with an Fc fragment or fused with human serum albumin.

12. The kinin derivative according to claim 8 is kinin conjugated with fatty acid or is a long-acting preparation of kinin.

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