WO2021253523A1 - 合成肽brap及其在制备新冠肺炎抗炎药中的应用 - Google Patents

合成肽brap及其在制备新冠肺炎抗炎药中的应用 Download PDF

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WO2021253523A1
WO2021253523A1 PCT/CN2020/100629 CN2020100629W WO2021253523A1 WO 2021253523 A1 WO2021253523 A1 WO 2021253523A1 CN 2020100629 W CN2020100629 W CN 2020100629W WO 2021253523 A1 WO2021253523 A1 WO 2021253523A1
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brap
synthetic peptide
lps
receptor
group
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PCT/CN2020/100629
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English (en)
French (fr)
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张万琴
李荫田
吉学文
赵丽美
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泰安市启航生物科技有限公司
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Priority to CA3134112A priority patent/CA3134112A1/en
Priority to JP2021552956A priority patent/JP7112792B2/ja
Priority to EP20911306.7A priority patent/EP3945093A4/en
Publication of WO2021253523A1 publication Critical patent/WO2021253523A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/18—Kallidins; Bradykinins; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of biomedicine, and relates to synthetic peptide brap (bradykinin receptor antagonism peptides, brap) and its application in preparing anti-acute lung injury, especially anti-inflammatory drugs for new coronary pneumonia.
  • ACE2 angiotensin-converting enzyme II
  • RAAS renin-angiotensin-aldosterone system
  • KKS Kallikrein-Kinin System
  • ACE2 angiotensin converting enzyme
  • the relative increase of ACE leads to the increase of AngII level, overactivating the AT1a receptor in the lungs, leading to increased pulmonary capillary permeability. Pulmonary edema appears subsequently, which aggravates the pulmonary inflammation; in the KKS system, Des-Arg bradykinin is an agonist of the BK1 receptor.
  • ACE2 has the ability to degrade Des-Arg bradykinin in the Des-Arg bradykinin-BK1 receptor pathway into inactive peptides. Decreased ACE2 levels lead to activation of the Des-Arg bradykinin-BK1 receptor pathway. BK1 receptor activation can promote inflammation.
  • the infected cells After virus infection, the infected cells produce inflammatory factors, which have the effect of anti-virus and regulating natural immune response.
  • the virus replicates in a large amount in the infected cells, it causes excessive activation of the immune system, and there is a significant increase in pro-inflammatory factors such as IL-6, TNF- ⁇ , IFN- ⁇ , and pro-inflammatory factors can activate and recruit other immune cells.
  • Immune cells can secrete more cytokines, rapid systemic immune inflammatory response, and cause multiple organ failure.
  • the onset of cytokine storm caused by new coronary pneumonia is still unclear.
  • Evidence has shown that the severity of severe new coronary pneumonia is closely related to the elevated level of the pro-inflammatory factor IL-6, which is an indicator of poor prognosis. Experts generally believe that the role of IL-6 inhibitors is worth looking forward to.
  • the present invention provides a synthetic peptide brap, and the amino acid sequence of the synthetic peptide brap is shown in SEQ ID No. 1.
  • the present invention also provides the application of the synthetic peptide brap in any of the following (1) to (4): (1) Preparation of drugs for inhibiting G protein coupling bradykinin B1 and B2 receptors; (2) Preparation of anti- Acute lung injury drugs; (3) Preparation of anti-inflammatory drugs for new coronary pneumonia; (4) Preparation of anti-allergic rhinitis drugs.
  • the drug administration mode of any one of (1) to (4) is intravenous administration or nasal administration.
  • the application of the present invention has the characteristics of low toxicity, and no toxicity is found in the limit experiment of the intravenous injection of 2000 mg/kg BW dose level.
  • the synthetic peptide brap disclosed in the present invention is a 10-peptide compound composed of 8 amino acids (Figure 1), and its amino acid sequence is shown in SEQ ID No. 1.
  • Brap can be industrially synthesized by solid-phase chemical synthesis technology, and the purity of synthetic peptide brap is ⁇ 99.5 ( Figure 2, 3).
  • Brap intravenous injection of 2000mg/kg BW dose level limit experiment showed no toxicity (Table 2).
  • ALI acute Lung Injury
  • COVID-19 coronavirus pneumonia
  • Acute lung injury is a diffuse respiratory disease characterized by leakage and inflammation of the lung parenchyma caused by damage to alveolar epithelial cells and capillary endothelial cells caused by multiple factors.
  • the pathogenic process of the lung includes the pathophysiological response of the virus to cells (pathophysiological response characterized by leakage and inflammation) and the immune pathological response of the body (the virus replicates in the infected cells, resulting in inflammatory factors
  • the virus is released, a large amount of virus replication leads to the excessive activation of the body's immune system and the damage caused by the cytokine storm.
  • the lung damage caused by the pathophysiological response of the virus to the cell is due to the new coronavirus corresponding target ACE2 belonging to the KKS and RAAS systems.
  • the KKS and RAAS systems are activated at the same time: in the RAAS system, the ACE2 level decreases, the balance between ACE2 and ACE is unbalanced, the relative increase of ACE, the increase of AngII level, and the excessive activation of the lung AT1a receptor , Resulting in increased pulmonary capillary permeability and pulmonary capillary leakage.
  • ACE2 also belongs to KKS, and Des-Arg bradykinin is a B1 receptor agonist of bradykinin (BK).
  • ACE2 has a degrading effect on Des-Arg bradykinin, making it an inactive peptide.
  • the Des-Arg bradykinin-BK1 receptor pathway is activated.
  • BK plays a biological role by binding to the receptor.
  • BK receptors are G protein coupled receptors (G protein coupled receptors, GPCRs), which mainly include two types, B1 and B2.
  • the B1 receptor of BK is inductively expressed, and tissue damage and inflammation can induce the expression of B1 receptor.
  • B1 receptor is activated to release pro-inflammatory cytokines, increase neutrophil penetration, activate neutrophils to produce excessive inflammatory mediators, reactive oxygen species (ROS), and participate in the inflammatory response at the injury site. Amplify inflammation and damage to the lungs. Activation of B2 receptors can significantly increase the permeability of microvessels, and a large amount of plasma components can leak out, causing lung leakage.
  • the present invention confirms that the synthetic peptide brap and the G protein coupled bradykinin B2 receptor have obvious target docking and inhibition effects. Brap and its target bradykinin B2 receptor are docked in an open pocket formed by the transmembrane helix of the B2 receptor protein. Residue No.
  • the invention also uses MDockPeP to connect the brap structure to the B1R of the BK.
  • Two different methods were used to model two B1 receptor structures, and they were docked with brap respectively. A total of 2 docking results were obtained, and the interaction between the B1 receptor (B1R) and the synthetic peptide brap (ligand) was further analyzed. Residues.
  • the present invention confirmed that brap binds in the receptor pocket in a U-shape ( Figure 8).
  • Intracellular calcium ion fluorescence technology was used to detect the effect of brap on the functional activity of Bradykinin B1 Receptor, which confirmed that brap has a significant inhibitory effect on B1 receptors and reduces excessive activation of B1 receptors (Figure 9).
  • the incubation period of new coronary pneumonia is the occurrence and development stage of the pathophysiological process of lung leakage and pneumonia caused by the reduction of ACE2 levels after the virus infection. From the incubation period to the diagnosis is the golden time to treat lung injury caused by new coronary pneumonia.
  • brap By antagonizing the G protein-coupled bradykinin B1 and B2 receptors, brap can effectively block the pathological response during the occurrence of new coronary pneumonia caused by the reduction of ACE2, and even through simple nasal drops, it can be significantly reduced
  • the pulmonary vascular permeability of guinea pigs caused by bradykinin is increased and lung leakage is significantly reduced.
  • the body's real elimination of the virus depends on the body's immune system to kill the virus.
  • the specific binding of the new coronavirus S protein to ACE2 on the cell is significant.
  • the virus starts to replicate in the cell, it stimulates the human immune system, and the infected cells produce inflammatory cytokines, which are anti-virus and immune Regulation effect.
  • the virus replicates in large numbers in the infected cells, it induces an excessive immune response in the body, and a cytokine storm occurs. Makes lung immune cells over-activated, producing a large number of cytokines.
  • LPS is a component of the cell wall of gram-negative bacteria and the main component of bacterial endotoxin. It can activate the mononuclear phagocyte system and make pro-inflammatory factors such as tumor necrosis factor (TNF- ⁇ ) and interleukin 6 (IL-6). ) The release and activation of neutrophils, inflammatory mediators and the production of reactive oxygen species (ROS) increase.
  • the present invention further uses lipopolysaccharide (LPS) to induce excessive inflammation and oxidative stress response and severe cytokine storm process in mice. It was found that after 6 hours of intraperitoneal injection of LPS (5mg/kg i.p.) into the mice, the mice in the LPS model group and the model administration group were both on the verge of death.
  • Intravenous injection of brap significantly reduces the level of endotoxin in the blood ( Figure 10), significantly reduces the overexpression of IL-6mRNA in lung tissue (Figure 11), and significantly reduces the levels of pro-inflammatory cytokines IL-6 and TNF- ⁇ in the blood ( Figure 12, 13), significantly reduced the excessively increased ROS content in lung tissue ( Figure 14-15), significantly reduced the pathological manifestations of lung injury (Figure 16).
  • brap nasal administration also significantly reduces the overexpression of IL-6 mRNA in lung tissue (Figure 11), reduces the levels of inflammatory factors IL-6 and TNF- ⁇ in the blood ( Figures 12, 13), and reduces The pharmacological effects of excessively increased ROS content in lung tissue ( Figure 14-15) and pathological changes such as lung interstitial inflammation, thickening of the pulmonary septum, and a large number of inflammatory cell infiltration (Figure 16) that significantly reduce lung injury.
  • IL-6 is an important inflammatory factor and an important pathway for inducing inflammatory storms.
  • the present invention found that intravenous and nasal administration of brap can significantly reduce the excessively elevated IL-6mRNA expression in the lung tissue of Balbc mice induced by LPS.
  • the present invention also found that not only the rat allergic nasal inflammation but also the BK-induced guinea pig pulmonary microvascular leakage has a significant inhibitory effect (P ⁇ 0.01), and both have a dose-response relationship ( Figures 6 and 7).
  • Nasal administration can quickly enter the blood circulation, quickly and significantly reduce lung leakage (Figure 7), lung injury (Figure 15), and significantly inhibit the expression of inflammatory factor IL-6mRNA in lung tissue (Figure 11).
  • Brap nasal administration can also be used to prevent and treat acute lung injury.
  • Brap is a small peptide synthesized using peptide solid-phase synthesis technology. It has been purified by high performance liquid chromatography (HPLC) and identified by mass spectrometry, providing quantitative data for brap's drug preparation, structural confirmation, and quality research.
  • brap has a blocking effect on both the B1 and B2 receptors of G protein-coupled BK
  • brap has a significant inhibitory effect on lung leakage during the development stage of new coronary pneumonia
  • brap has an effect on excessive inflammation and
  • the oxidative stress response has a significant inhibitory effect and a significant protective effect on lung tissue damage; if the disease progresses to a critical inflammatory storm stage, the synthetic peptide brap will significantly inhibit the over-activated immune inflammatory response and reduce the over-activation of the body's immune system.
  • different routes of administration can be used in the nasal cavity or intravenously.
  • glucocorticoids are mainly used in clinical anti-inflammatory treatments, and glucocorticoids have obvious side effects.
  • Figure 1 is the amino acid composition analysis of the synthetic peptide brap;
  • A is the amino acid composition curve diagram of the synthetic peptide brap drawn by the amino acid analyzer;
  • B is the amino acid composition diagram of the standard product used in the synthetic peptide brap amino acid detection.
  • Figure 2 shows the results of synthetic peptide brap high performance liquid chromatography.
  • Figure 3 shows the mass spectrum result of the synthetic peptide brap.
  • Figure 4 shows the target docking test of the synthetic peptide brap to the G protein-coupled bradykinin B2 receptor.
  • Figure 5 shows the inhibitory effect of synthetic peptide brap on bradykinin B2 receptor
  • A is the dose-response curve of the positive control bradykinin B2 receptor inhibitor HOE140 of synthetic peptide brap
  • B is the synthetic peptide brap on bradykinin B2 Dose-response curve of receptor inhibition.
  • Figure 6 shows the pharmacodynamic effect of synthetic peptide brap nasal administration on allergic nasal inflammation in rats, A is the score of allergic rhinitis, and B is the change of score before and after treatment.
  • Figure 7 shows the nasal administration of the synthetic peptide brap significantly inhibits bradykinin-induced pulmonary microvascular leakage in guinea pigs.
  • A is the standard curve of OD value-EB concentration, and B is the EB content of lung tissue.
  • Figure 8 shows the target docking of the synthetic peptide brap to the G protein-coupled bradykinin B1 receptor.
  • Figure 9 shows the inhibitory effect of the synthetic peptide brap on the bradykinin B1 receptor;
  • A is the dose-response curve of the bradykinin B1 receptor antagonist R892;
  • B is the dose of the synthetic peptide brap on the bradykinin B1 receptor inhibition -Response curve.
  • Figure 10 shows that brap significantly reduces the significant increase in blood endotoxin levels induced by LPS.
  • Figure 11 shows that brap significantly reduces LPS-induced lung tissue.
  • Figure 12 shows that brap significantly reduces the increase in the level of IL-6 in the blood induced by LPS
  • A is lipopolysaccharide LPS promotes the release of inflammatory cytokine IL-6
  • B is the synthetic peptide brap induced LPS (5mg/kg ip) 6h
  • C is the effect of synthetic peptide brap on the release of inflammatory cytokine IL-6 induced by LPS 12h.
  • FIG. 13 shows that brap significantly reduces the increase of TNF- ⁇ in the blood induced by LPS.
  • A is lipopolysaccharide LPS that promotes a significant increase in the content of pro-inflammatory cytokine TNF- ⁇ ;
  • B is the inflammatory cytokine induced by synthetic peptide brap on LPS 6h The effect of TNF- ⁇ release;
  • B is the effect of synthetic peptide brap on the release of inflammatory cytokine TNF- ⁇ induced by LPS 12h.
  • Figure 14 is a qualitative analysis of brap significantly reducing the increase in ROS content in lung tissue induced by LPS.
  • Figure 15 is a quantitative analysis of brap significantly reducing the increase in ROS content in lung tissue induced by LPS.
  • Figure 16 shows that brap significantly protects lung injury induced by LPS;
  • A is the normal control group
  • B is the LPS model group
  • C is the sp2 group
  • D is the brap intravenously administered high-dose group
  • E is the brap intravenously administered middle-dose group
  • F is Brap intravenous administration low-dose group
  • G is brap nasal administration group
  • H is model dexamethasone group (positive control group).
  • step b sequentially add different amino acids in the sequence
  • the crude product obtained was purified by high performance liquid chromatography, the peptide was lyophilized, and the peptide was detected, and then mass spectrometry was used for product identification.
  • the high performance liquid chromatography is shown in Figure 2
  • the purity of the obtained synthetic peptide brap is 99.72%
  • the mass spectrum is shown in Figure 3
  • the theoretical molecular weight of the synthetic peptide brap is 1023.17, and the molecular weight is correct as identified by the mass spectrum.
  • Example 3 The docking and binding mode of Bradykinin B2 receptor (B 2 R) target and brap
  • NCBI Blast was used to expand the B2 receptor (B2R) of Bradykinin (BK) based on the sequence alignment of the PDB library, and 5UNF with better sequence similarity and coverage percentage was selected as the template.
  • B2R B2 receptor
  • BK Bradykinin
  • 5UNF 5UNF with better sequence similarity and coverage percentage was selected as the template.
  • homology modeling module Prime in the Schrodinger software package to model the three-dimensional structure of B2R. According to the different parameter settings of the model, two B2R structure files were obtained. In order to ensure the rationality and repeatability of subsequent docking, they are knowledge-based receptor structure and energy-based receptor structure.
  • MDockPeP docked the synthetic peptide brap to the two receptor structures and performed a total of 2 docking experiments. In the docking experiment, the binding position of the synthetic peptide brap is distributed in the middle of the open pocket composed of the transmembrane helix of the receptor protein ( Figure 4).
  • Example 4 Using intracellular calcium ion fluorescence technology to detect the inhibitory effect of brap on Bradykinin B2 Receptor
  • HEK293/G15/Bradykinin2 (B 2 R) experimental method use intracellular calcium ion fluorescence technology to detect the inhibitory effect of synthetic peptide brap on Bradykinin B2 Receptor
  • the HEK293/G15/Bradykinin2 cells whose growth confluence reached 80% were digested with trypsin and counted, and plated at a density of 2 ⁇ 10 4 cells/mL per well on a 384-well cell culture plate coated with matrigel in advance. middle;
  • EC10, EC20 and EC80 represent different stimulation intensities to B2 receptors
  • test curve is smooth, a clear S-shaped curve, and IC50 is about 1mM.
  • Modeling of SD rats sensitization by intraperitoneal injection and stimulation by nasal drip.
  • ovalbumin 1 mg ovalbumin (Class V Sigma, USA) was dissolved in 1 ml of normal saline, and 30 mg of aluminum hydroxide was added as an immune adjuvant.
  • Each rat was intraperitoneally injected with the ovalbumin suspension prepared above with 1ml/time every other day, a total of 7 times.
  • the ovalbumin nasal cavity was challenged, and 20mg ovalbumin (Class V Signa, USA) was dissolved in Make a 2% solution in 1ml of normal saline, and use this 2% ovalbumin solution for nasal instillation to challenge.
  • Each rat is instilled 50ul of nasal cavity for 7 consecutive days.
  • the blank control group was injected intraperitoneally with 30 mg of aluminum hydroxide + 1 ml of physiological saline, and the method was the same as above. On the 15th day, the nose was instilled with normal saline.
  • Administration method administer the drug with a small amount of sample, administer to the rat's bilateral nasal cavity, 50 ⁇ L/each side, 20min after the administration, give the rat's bilateral nasal cavity 2% ovalbumin solution, 50 ⁇ L/each side Nasal drops are used for provocation; after the above treatments continue for 4 weeks, the efficacy of the drugs is observed.
  • Synthetic peptide brap high dose Take the synthetic peptide brap freeze-dried powder (5mg/bottle), add 1.63ml sterile normal saline to dissolve;
  • Synthetic peptide brap medium dose take the synthetic peptide brap freeze-dried powder (5mg/bottle), add 4.89ml sterile normal saline to dissolve;
  • the concentration of the synthetic peptide sp2 high dose group is the same as the synthetic peptide brap high dose group, and the concentration of the synthetic peptide sp2 medium dose group is the same as the synthetic peptide brap middle dose.
  • the groups are the same.
  • the amino acid sequence of synthetic peptide sp2 is shown in SEQ ID No.2.
  • FIG. 6A shows the efficacy scores of synthetic peptide brap on rats with allergic rhinitis.
  • the experimental results show that compared with the model group, the synthetic peptide brap high-dose, medium-dose and low-dose groups can improve The symptoms of allergic rhinitis induced by OVA, and the scores have significant differences, suggesting that the synthetic peptide brap has a good therapeutic effect on allergic rhinitis.
  • Figure 6B shows the changes in the scores of rats before and after treatment.
  • Example 6 The effect of nasal administration of synthetic peptide brap on bradykinin-induced pulmonary microvascular leakage in guinea pigs
  • Normal control group intravenous injection of an equal volume of saline on the lateral side of the foot for 3 consecutive days;
  • Model control group intravenous injection of an equal volume of saline on the lateral side of the foot for 3 consecutive days;
  • Positive control group intravenous injection on the lateral side of the foot, dexamethasone 1.25mg/3ml/kg BW for 3 consecutive days;
  • Synthetic peptide brap high-dose group inject a synthetic peptide brap with a concentration of 3.0mM/L into the nasal cavity of guinea pigs with a micropipette gun, 50 ⁇ l/each side/day, for 3 consecutive days;
  • Synthetic peptide brap middle dose group use a micropipette gun to inject a synthetic peptide brap with a concentration of 1.0mM/L into the nasal cavity of guinea pigs, 50 ⁇ l/each side/day, for 3 consecutive days;
  • Low-dose synthetic peptide brap group Inject the synthetic peptide brap with a concentration of 0.33mM/L into the nasal cavity of guinea pigs with a micropipette gun, 50 ⁇ l/each side/day, for 3 consecutive days.
  • Lung tissue EB determination 30 minutes after injection of bradykinin, the animals were anesthetized and sacrificed by taking blood from the carotid artery, performing blood routines, opening the chest cavity, cutting the right ventricle and left atrium, and pulmonary artery cannulation, and lavage the pulmonary circulation with 30mL saline , Until the effluent is clear. Take out the lower lobe of the right lung, rinse the surface blood with normal saline, and absorb the water on the filter paper; take 100 mg and cut it into 2 mL formamide solution, and incubate it in a water bath at 45°C for 24 hours.
  • EB content of lung tissue is calculated from the EB standard curve (the EB standard curve is shown in Fig. 7A), as shown in Fig. 7B.
  • Evans Blue can be combined with albumin.
  • the exudation of EB reflects the exudation of protein.
  • it reflects the permeability of lung microvascular and the degree of pulmonary microvascular leakage.
  • the lung tissue EB content of the model group increased significantly (** means P ⁇ 0.01); the low, medium, and high-dose synthetic peptide brap groups were significantly lower than the model group (P ⁇ 0.01), with a dose-response relation.
  • ⁇ indicates, P>0.05
  • two B1 structure files were obtained, namely the knowledge-based receptor structure and the energy-based receptor structure.
  • the sequence of the B1 receptor is downloaded from the uniprot library, and the sequence of the B1 receptor used in the homology modeling is shown in SEQ ID No.7.
  • MDockPeP is used to dock the structure of B1 receptor protein and brap.
  • Brap is a 10-peptide compound composed of 8 amino acids.
  • the green in the figure represents the receptor protein molecule, and the light blue represents the synthetic peptide brap molecule.
  • the green in the figure represents the receptor protein molecule, and the light blue represents the brap molecule.
  • the synthetic peptide brap binds to the receptor pocket in a U-shape.
  • Example 8 The inhibitory effect of synthetic peptide brap on bradykinin B1 receptor
  • the HEK293/G ⁇ 15/B1 cells that need to be resuscitated were quickly taken out of the liquid nitrogen tank and melted in a 37°C water bath. Quickly add the cell suspension to the pre-warmed DMEM+10% FBS medium, put it in a centrifuge, and centrifuge for 5 minutes at 1000 rpm. Take out the centrifuge tube, discard the supernatant, add fresh pre-warmed culture medium to the centrifuge tube, resuspend the cells, add the cell suspension to a culture dish, and incubate at 37°C with 5% CO 2 .
  • HEK293/G ⁇ 15/B1 cells stably expressing the B1 receptor were cultured in DMEM+10% FBS; cell culture conditions: HEK293/G ⁇ 15/B1 cell line was cultured routinely, containing 10% fetal bovine serum and passaged in DMEM.
  • the cells When the cells are 80-90% full of the culture dish, the cells are trypsinized with 0.25% trypsin, and the cells are resuspended in a new medium, and the cells are passaged in an appropriate ratio, approximately once every 2 to 3 days.
  • the HEK293/G15/Bradykinin1 cells whose growth confluence reached 80% were digested with trypsin and counted, and plated on the 384-well cells that had been coated with matrigel in advance at a density of 2 ⁇ 10 4 cells/mL. In the culture plate.
  • the final concentration of EC10 is 0.3nM
  • the final concentration of EC20 is 0.6nM
  • the final concentration of EC80 is 18nM.
  • brap and EC80 work together to have an obvious S-shaped curve, indicating that it has a significant inhibitory effect on the excessive activation of B1 receptors; the effect of compounds that work together with EC10 and EC20 is not obvious, and it is speculated that too much calcium ions have been released in the previous reaction. , Can not cause secondary signals in a short time.
  • Feeding conditions SPF aseptic breeding environment, 25 degrees constant temperature environment, 12h alternating light, adequate food and water supply.
  • Grouping and processing randomly divided into 8 groups (A-H), 6 per group;
  • a Normal control group injection of saline via tail vein once a day;
  • Model administration sp2 group tail vein injection of sp2 (16mg/kgBW) once a day;
  • Model administration brap low-dose group intravenous injection of brap (4mg/kgBW) once a day;
  • Model administration brap nasal administration group brap (3mM), nasal administration on both sides, 15ul/each side; 1 time/day;
  • Model dexamethasone group (positive control group): dexamethasome, DEX, 5mg/kg intraperitoneal injection, once a day.
  • Transparent xylene 10min each for xylene I and II;
  • Model group The pulmonary septum is thickened, inflammatory cell infiltration, and the pulmonary septum is focally fused with each other. There were no abnormalities in the high-dose brap intravenous administration group (D) and the brap nasal cavity administration group (G).
  • Figure 16B shows the comparison between the LPS model group and the normal control group.
  • Figure 16A shows that the alveolar septum is thickened, inflammatory cell infiltration, and the lung septum has focal mutual fusion;
  • Figure 16C is the LPS + synthetic peptide sp2 group, with obvious inflammatory cell infiltration;
  • Figure 16D is LPS In the high-dose +brap group, there was no thickening of the alveolar septum and no obvious inflammatory cell infiltration;
  • E and F groups were the medium and low-dose groups of LPS+brap, respectively.
  • G group It is the nasal administration group, similar to the D group, without obvious abnormalities;
  • H group is the dexamethasone positive control group, with obvious inflammatory cell infiltration, and no focal fusion of the pulmonary septum.
  • the concentration gradient of endotoxin standard is 1.0, 0.5, 0.25 and 0.1 EU/ml;
  • 1.1 Lung tissue samples Take fresh lung tissue samples of each group of mice constructed in Example 9 and quickly freeze them with liquid nitrogen as soon as possible, and store them at -80°C.
  • brap nasal drip group Can reduce LPS, 12h blood TNF-a and IL-6 levels, P ⁇ 0.05.
  • Example 13 Flow cytometry to detect the fluorescence intensity of ROS in lung tissues of each group
  • Figure 14A is the normal control group
  • Figure 14B is the LPS model group (5mg/kgBW intraperitoneal injection)
  • Figure 14C is LPS+16mg/kgBW synthetic peptide brap
  • Figure 14D is LPS+8mg/kgBW synthetic Peptide brap
  • Figure 14E is LPS+4mg/kg BW synthetic peptide brap
  • Figure 14F is LPS+ synthetic peptide brap (injected into the nasal cavity on both sides, 15 ⁇ L/each side/day)
  • Figure 14G is the positive control group (LPS+dexamethasone 5mg/kg Intraperitoneal injection).
  • Figure 15 shows the average fluorescence intensity of ROS in the lung tissue of each group of mice detected by flow cytometry.
  • Test sample synthetic peptide brap
  • the sample is accurately divided, no need to weigh, 5mg/bottle x 8 bottles (for the first mouse), 8mg/bottle x 20 bottles (for the other 4 mice);
  • Preparation method Take out the synthetic peptide brap freeze-dried powder from -20 degrees, restore it to room temperature for 15 minutes, add 1.5ml of saline, shake, and completely dissolve.
  • Dosing volume mouse tail vein injection volume with the maximum volume of 0.5ml/each time, slow injection
  • test substance synthetic peptide brap

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Abstract

合成肽brap及其在制备新冠肺炎抗炎药中的应用，属于生物医药技术领域。一种合成肽brap，其氨基酸序列如SEQ ID No.1所示。brap对G蛋白偶联的缓激肽B1和B2受体均有明显的靶点对接和抑制作用；brap鼻腔给药具有对过敏性鼻部炎症的局部作用和对肺渗漏、肺损伤及LPS诱发的细胞因子风暴的全身性药效作用。brap静脉注射对LPS诱发小鼠出现的过度的炎症和氧化应激反应以及严重肺损伤均有明显抑制作用，对LPS诱发的炎性因子风暴中促炎因子IL-6、TNF-α的过度释放和IL-6 mRNA的过表达以及活性氧自由基(reactive oxygen species，RSO)大量产生均有明显抑制作用。

Description

合成肽brap及其在制备新冠肺炎抗炎药中的应用 技术领域
本发明属于生物医药技术领域，涉及合成肽brap(bradykinin receptor antagonism peptides，brap)及其在制备抗急性肺损伤特别是新冠肺炎抗炎药中的应用。
背景技术
严重的新型冠状病毒肺炎(COVID-19)正严重危胁人类的生命。科学工作者发现新冠病毒对应的靶点是血管紧张素转化酶Ⅱ(ACE2)。当病毒的S蛋白与细胞上的ACE2特异性结合时，病毒进入细胞，ACE2是新冠病毒感染细胞的靶点(即细胞受体)，也是感染后导致肺损伤病理发生的关键因素。病毒在被感染的细胞内复制并引起ACE2水平降低，ACE2是肾素-血管紧张素-醛固酮系统(RAAS)也是激肽释放酶-激肽系统(Kallikrein-Kinin System，KKS)的重要成员。ACE2水平降低，引起RAAS系统中，ACE2和ACE(血管紧张素转化酶)失衡，ACE相对增强，导至AngⅡ水平升高，过度激活肺部AT1a受体，导致肺部毛细血管通透性增加，随之出现肺水肿，加重肺部炎症反应；在KKS系统中，Des-Arg缓激肽是BK1受体的激动剂。ACE2具有将Des-Arg缓激肽-BK1受体途径中的Des-Arg缓激肽降解为无活性作用的肽。ACE2水平降低导致Des-Arg缓激肽-BK1受体途径激活。BK1受体激活具有促进炎症作用。
病毒感染后，受感染的细胞产生炎性因子，具有抗病毒和调控天然免疫应答作用。但是当病毒在被感染细胞内大量复制，引起免疫系统过度激活，出现IL-6、TNF-α、IFN-γ等促炎因子的显著升高，促炎因子可以激活和招暮其它免疫细胞，免疫细胞可以分泌更多的细胞因子，迅速出现全身免疫炎症反应，引起多器官功能衰竭。目前，新冠肺炎所致细胞因子风暴发病过程尚不明确。己有证据表明，重症新冠肺炎的严重程度与促炎因子IL-6的升高水平密切相关，IL-6升高是病情预后不良的一个指标。专家普遍认为IL-6抑制剂的作用值得期待。
发明内容
本发明提供了一种合成肽brap，所述合成肽brap的氨基酸序列如SEQ ID No.1所示。
本发明还提供了合成肽brap的如下(1)～(4)中任一所述的应用：(1)制备抑制G蛋白偶联的缓激肽B1和B2受体药物；(2)制备抗急性肺损伤药物；(3)制备新冠肺炎抗炎药物；(4)制备抗过敏性鼻炎药物。
进一步地，上述技术方案中，(1)～(4)中任一所述的药物的给药方式为静脉给药或鼻腔给药。
本发明所述的应用，具有毒性低的特征，静脉注射2000mg/kgBW剂量水平的限度实验未见毒性。
本发明公开的合成肽brap是由8种氨基酸组成的10肽化合物(图1)，其氨基酸序列如SEQ ID No.1所示。brap可用固相化学合成技术进行工业化合成，合成肽brap的纯度≥99.5(图2、3)。brap静脉注射2000mg/kg BW剂量水平的限度实验未见毒性(表2)。最近brap与急性肺损伤(Acute Lung Injury，ALI)特别是新型冠状病毒肺炎(COVID-19))的关系被发现。急性肺损伤是多种因素导致的肺泡上皮细胞和毛细血管内皮细胞损伤造成的以渗漏和炎症为特征肺实质弥漫性的呼吸系统疾病。新冠病毒进入人体，对肺的致病过程包括病毒对细胞的病理生理反应(渗漏和炎症为特征的病理生理反应)和机体免疫病理反应(病毒在受感染的细胞内复制，导致炎性因子释放，病毒大量复制导致机体免疫系统过度激活，出现细胞因子风暴)造成的损伤。
病毒对细胞的病理生理反应造成的肺损伤是由于新冠病毒对应的靶点ACE2同属于KKS和RAAS系统。当病毒感染导致ACE2水平降低时，同时激活了KKS和RAAS系统：在RAAS系统，ACE2水平降低、ACE2和ACE之间的平衡失衡、ACE相对增强、AngⅡ水平升高、过度激活肺部AT1a受体，导致肺部毛细血管通透性增加、肺微血管渗漏。ACE2也同属于KKS，Des-Arg缓激肽是缓激肽(Bradykinin，BK)的B1受体激动剂。ACE2对Des-Arg缓激肽具有降解作用，使之成为无活性作用的肽。当新冠病毒感染引起被感染细胞ACE2水平降低，导致Des-Arg缓激肽-BK1受体途径激活。BK通过与受体结合发挥生物学作用。BK受体为G蛋白偶联受体(G protein coupled receptor，GPCR)主要包括B1和B2两种类型。BK的B1受体为诱导性表达，组织损伤及炎症反应可诱导B1受体表达。B1受体被激活，促炎性细胞因子释放、增加中性粒细胞渗透、激活中性粒细胞产生过多的炎症介质、氧自由基(reactive oxygen species，ROS)，参与损伤部位的炎症反应，放大肺部炎症与损伤。B2受体被激活可明显增加微血管的通透性，血浆成分大量渗出，引起肺渗漏。本发明证实合成肽brap与G蛋白偶联的缓激肽B2受体有明显的靶点对接和抑制作用。brap与其靶点缓激肽B2受体对接分布在B2受体蛋白的跨膜螺旋组成的张口的口袋中，1号残基指向口袋外部，10号残基位于口袋底部位。采用brap与B2受体结合部位分析，10个残基均与B2受体对接，並且计算了小肽每个残基的能量供献(图4)。本发明证实功能上，brap对B2受体有明显抑制作用(图5)。本发明证实，双侧鼻腔滴入合成肽brap(50μl/每侧)不仅 对大鼠过敏性鼻部炎症而且对BK诱发的豚鼠肺微血管渗漏具有明显抑制作用(P<0.01)，均具有剂量-反应关系(图6、7)。本发明同时采用MDockPeP进行brap结构对BK的B1R进行的对接。用两个不同方法，模建了两种B1受体结构，分别与brap进行对接，共获得2次对接结果，并进一步解析了B1受体(B1R)与合成肽brap(配体)的相互作用残基。本发明证实brap以U型结合于受体口袋中(图8)。用胞内钙离子荧光技术检测brap对Bradykinin B1 Receptor的功能活性的影响，证实brap对B1受体具有明显抑制作用、降低B1受体过度激活(图9)。新冠肺炎的潜伏期，正是病毒感染后，ACE2水平降低导致肺渗漏和肺炎症的病理生理过程的发生、发展阶段。从潜伏期到确诊是治疗新冠肺炎肺损伤的黄金时间。brap通过对G蛋白偶联的缓激肽B1和B2受体的拮抗作用，有效地阻断ACE2降低所致新冠肺炎发生过程中的病理反应，甚至通过简单的滴鼻给药，就可明显减轻缓激肽引起的豚鼠肺血管通透性增加、明显减轻肺渗漏。
然而，机体真正清除病毒，是依靠人体的免疫系统对病毒的杀灭。在感染初期，显著新冠病毒S蛋白与细胞上的ACE2的特异性结合，当病毒在细胞内开始复制的时候，刺激了人体免疫系统，被感染的细胞产生炎性细胞因子，具有抗病毒和免疫调节作用。感染后期 ,当病毒在被感染细胞内大量复制，诱发机体的过度免疫反应，出现细胞因子风暴。使得肺部免疫细胞过度活化，产生大量细胞因子。
LPS是革兰氏阴性菌细胞壁成分，也是细菌内毒素的主要成分，能够激活单核呑噬细胞系统，使促炎性因子如肿瘤坏死因子(TNF-α)、白细胞介素6(IL-6)释放、激活中性粒细胞、炎性介质和氧自由基(reactive oxygen species，ROS)的产生增多。本发明进一步，采用脂多糖(lipopolysaccharide，LPS)诱发小鼠出现的过度的炎症和氧化应激反应以及严重的细胞因子风暴过程。结果发现给小鼠腹腔注射LPS(5mg/kg i.p.)6h后，LPS模型组和模型给药组小鼠均表现濒临死亡的状态。精神萎靡、呼吸急促、耸毛、稀便、眼周出现分泌物和流泪等全身多器官受累症状。血清细菌内毒素、IL-6、TNF-α含量及肺组织IL-6 mRNA表达均显著升高、肺部ROS含量也显著升高(图10-15)，并且出现肺损伤的病理表现:肺间隔增厚、炎细胞浸润、肺间隔有局灶性相互融合(图16)。
brap静脉注射明显降低血液中内毒素水平(图10)、明显降低肺组织中IL-6mRNA的过表达(图11)、明显降低血液中促炎症的细胞因子IL-6和TNF-α的水平(图12、13)、明显降低肺组织中过度增加的ROS含量(图14-15)、明显减轻肺损伤的病理表现(图16)。本发明同时发现brap鼻腔给药也具有明显降低肺组织中IL-6 mRNA的过表达(图11)、 降低血液中炎性因子IL-6和TNF-α的水平(图12、13)、降低肺组织中过度增加的ROS含量(图14-15)和明显减轻肺损伤的肺间质性炎症、肺间隔增厚及大量炎性细胞浸润等病理改变(图16)的药效作用。
己有报道，新冠肺炎重型患者易出现细胞因子风暴(即炎症风暴)。科学工作者发现IL-6是重要的炎性因子，是诱发炎症风暴的重要通路。本发明发现，静脉和鼻腔给予brap可明显降低LPS诱发的Balbc小鼠过度升高的肺组织IL-6mRNA表达。本发明还发现不仅对大鼠过敏性鼻部炎症而且对BK诱发的豚鼠肺微血管渗漏具有明显抑制作用(P<0.01)，均具有剂量-反应关系(图6、7)。鼻腔给药可迅速进入血液循环，迅速明显减轻肺渗漏(图7)、肺损伤(图15)，明显抑制炎性因子IL-6mRNA在肺组织的表达(图11)。提供了brap鼻腔给药也可用于防治急性肺损伤的实验依据。brap是釆用多肽固相合成技术合成的小肽，经高效液相色谱(HPLC)纯化、质谱分析鉴定，为brap的药物制备、结构确证、质量研究等方面提供了量化资料。由于brap对G蛋白偶联的BK的B1和B2受体均具有阻断作用，在新冠肺炎发生发展阶段，brap对肺渗漏具有明显的抑制作用；在炎症进展阶段，brap对过度的炎症和氧化应激反应具有明显抑制作用以及对肺组织损伤的显著的保护作用；如果病情发展到危重的炎症风暴阶段，合成肽brap明显抑制过度激活的免疫炎症反应、降低机体免疫系统的过度激活。而且可以根据病情进展，采用鼻腔或静脉不同途径给药。目前临床上用于抗炎治疗的药物主要是糖皮质激素，糖皮质激素具有明显的副作用。现有技术中，未见有类似的化合物在新冠肺炎抗炎药物中的应用。
附图说明
图1为合成肽brap的氨基酸组成分析；A为由氨基酸分析仪绘制的合成肽brap的氨基酸组成曲线图；B为合成肽brap氨基酸检测时所用标准品的氨基酸成分图。
图2为合成肽brap高效液相色谱结果。
图3为合成肽brap质谱结果。
图4为合成肽brap对G蛋白偶联的缓激肽B2受体的靶点对接试验。
图5为合成肽brap对缓激肽B2受体的抑制作用；A为合成肽brap的阳性对照缓激肽B2受体抑制剂HOE140的剂量-反应曲线；B为合成肽brap对缓激肽B2受体抑制作用的剂量-反应曲线。
图6为合成肽brap鼻腔给药对大鼠过敏性鼻部炎症的药效作用，A为过敏性鼻炎评分，B为治疗前后评分变化。
图7为合成肽brap鼻腔给药明显抑制缓激肽诱导的豚鼠肺微血管渗漏，A为OD值-EB浓度标准曲线，B为肺组织EB含量。
图8为合成肽brap对G蛋白偶联的缓激肽B1受体的靶点对接。
图9为合成肽brap对缓激肽B1受体的抑制作用；A为缓激肽B1受体拮抗剂R892的剂量-反应曲线；B为合成肽brap对缓激肽B1受体抑制作用的剂量-反应曲线。
图10为brap明显降低LPS诱发的血液中内毒素水平的显著升高。
图11为brap明显降低LPS诱发的肺组织.IL-6 mRNA的过度表达
图12为brap明显降低LPS诱发的血液中内IL-6水平的升高，A为脂多糖LPS促使炎性细胞因子IL-6释放；B为合成肽brap对LPS(5mg/kg i.p.)6h诱发的炎性细胞因子IL-6释放的影响；C为合成肽brap对LPS 12h,诱发的炎性细胞因子IL-6释放的影响。
图13为brap明显降低LPS诱发的血液中TNF-α的升高，A为脂多糖LPS促使促炎性细胞因子TNF-α含量显著增加；B为合成肽brap对LPS 6h诱发的炎性细胞因子TNF-α释放的影响；B为合成肽brap对LPS 12h诱发的炎性细胞因子TNF-α释放的影响。
图14为brap明显降低LPS诱发的肺组织中ROS含量的升高定性分析。
图15为brap明显降低LPS诱发的肺组织中ROS含量的升高定量分析。
图16为brap明显保护LPS诱发的肺损伤；A为正常对照组、B为LPS模型组、C为sp2组、D为brap静脉给药高剂量组、E为brap静脉给药中剂量组F为brap静脉给药低剂量组G为brap鼻腔给药组、H为模型给地塞米松组(阳性对照组)。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1合成肽brap的氨基酸组成试验
准确称取10mg的样品于水解管中，加入6mol/L的盐酸20ml，真空处理脱气，冲入氮气，封管。110℃水解22-24h，冷却后用去离子水定容至50mL，混匀。准确取1mL水解液，真空干燥，加1mL去离子水蒸干，再加1mL去离子水蒸干，准确加入1mL0.02mol/L盐酸复溶。用0.22μm的滤膜过滤上机(日立L-8900氨基酸分析仪)测试。测试结果如表1和图1所示。
表1合成肽brap的氨基酸组成及含量
Figure PCTCN2020100629-appb-000001
Figure PCTCN2020100629-appb-000002
由表1和图1可知，合成肽brap由8种氨基酸组成
实施例2合成肽brap的合成过程、HPLC纯化和质谱鉴定
本技术由苏州强耀生物科技有限公司提供。
合成顺序：从序列C端到N端，步骤如下：
a.称取n当量树脂(固相合成载体)放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF、DCM(适量是指以可使树脂充分鼓动起来为宜)，DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；
b.往反应器中加入序列中第二个氨基酸(也为2n当量)，2n当量HBTU(1－羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA，N 2鼓泡反应半小时，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测；
c.依步骤b的方式依次加入序列中不同的氨基酸；
d.将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA，2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂；
e.得到滤液，然后向滤液中加入大量乙醚，析出粗产物，然后离心，清洗即可得 到SEQ ID No.1所示序列的粗产物
用高效液相色谱将所得粗品提纯、多肽冻干、多肽检测，然后使用质谱进行产物鉴定。高效液相色谱如图2所示，所得合成肽brap的纯度为99.72％；质谱如图3所示，合成肽brap的理论分子量是1023.17，质谱鉴定分子量正确。
实施例3 Bradykinin B2 receptor(B 2R)靶点与brap的对接及结合模式
本技术由南京欧际医药科技服务有限公司提供。
采用NCBI Blast对Bradykinin(BK)的B2 receptor(B2R)展开基于PDB库的序列比对，选取了序列相似性和覆盖百分比较好的5UNF为模板。采用Schrodinger软件包中的同源模建模块Prime对B2R进行了三维结构模建。根据模建的参数设置不同获得了两种B2R的结构文件，为确保后续对接的合理性和可重复性，分别是基于知识的受体结构和基于能量的受体结构。进一步采用，MDockPeP将合成肽brap对接到两种受体结构上，总共进行了2次对接实验。在对接实验中，合成肽brap的结合位置分布在受体蛋白跨膜螺旋组成的张开的口袋中间(图4)。
实施例4用胞内钙离子荧光技术检测brap对Bradykinin B2 Receptor的抑制作用
本技术由武汉合研生物医药科技有限公司提供。
HEK293/G15/Bradykinin2(B 2R)实验方法:用胞内钙离子荧光技术检测合成肽brap对Bradykinin B2 Receptor的抑制作用
1.将生长汇合度达到80％的HEK293/G15/Bradykinin2细胞用胰酶消化后计数，按照每孔2x10 4个/mL的密度铺于提前包被过matrigel的黑边透明的384孔细胞培养板中；
2.将铺好的384孔细胞培养板放入5％CO 2 37℃培养箱中过夜培养；
3.实验当天将化合物合成肽brap用HBSS溶解成30mM的储备液；
4.每孔加入10uL 4X免洗Fluo8染料，室温孵育1小时；。
5.在细胞孵育的过程中，用含有0.1％BSA的HBSS，将待测化合物合成肽brap进行五倍向下稀释。阳性抑制剂HOE140起始浓度为3uM；
6.EC10、EC20和EC80代表对B2受体的不同刺激强度；
7.将配好的待测化合物加到细胞培养板中,放入FLIPR中，进行数据记录。
如图5结果显示，测试曲线平滑，是一个明显的S型曲线，IC50是在1mM左右。
实施例5合成肽brap鼻腔给药对大鼠过敏性鼻部炎症的药效实验
本技术由上海美轩生物科技有限公司提供。
一.动物模型制备
1.造模方法:
SD大鼠造模：腹腔注射致敏与鼻腔滴注激发两个阶段。卵清蛋白腹腔注射，以1mg卵清蛋白(V级Sigma，美国)溶于1ml生理盐水，30mg氢氧化铝加入为免疫佐刘。每只大鼠用上述所制卵清蛋白混悬液腹腔注射1ml/次隔日一次，共7次，第15天进行卵清蛋白鼻腔激发，以20mg卵清蛋白(V级Signa，美囯)溶于1ml的生理盐水中，制成2％的溶液，用此2％卵清蛋白溶液滴鼻激发，每只大鼠每测鼻腔50ul滴鼻，连续7天。
空白对照组仅以氢氧化铝30mg+生理盐水1ml混匀后腹腔注射，方法同上述。第15天用生理盐水滴鼻。
2.症状评定
最后一次滴鼻处理后，观察，根据评分标准表对各只大鼠进行评定:喷嚏、抓鼻、喘息、鼻分泌物
二、动物模型治疗及评价
1、给药方法：用微量加样抢给予药物，大鼠双侧鼻腔给药，50μL/每侧，给药20min后，大鼠双侧鼻腔给2％卵清蛋白溶液，50μL/每侧进行滴鼻激发；上述处理连续4周后，观察药效。
2、药物配置
合成肽brap高剂量：取合成肽brap冻干粉(5mg/瓶)，加入1.63ml无菌生理盐水溶解；
合成肽brap中剂量：取合成肽brap冻干粉(5mg/瓶)，加入4.89ml无菌生理盐水溶解；
合成肽brap低剂量：取合成肽brap冻干粉(5mg/瓶)，加入14.67ml无菌生理盐水溶解；
同时设置合成肽sp2高剂量组和合成肽sp2中剂量组为对照组，合成肽sp2高剂量组的浓度与合成肽brap高剂量组相同，合成肽sp2中剂量组的浓度与合成肽brap中剂量组相同。合成肽sp2的氨基酸序列如SEQ ID No.2所示。
3、症状评定
最后一次OVA激发后，对大鼠进行评分。实验结果如图6所示，图6A为合成肽brap对过敏性鼻炎大鼠的药效评分，实验结果表明，和模型组相比，合成肽brap高剂 量、中剂量和低剂量组均可以改善由OVA诱导的过敏性鼻炎症状，且评分具有显著差异性，提示合成肽brap对过敏性鼻炎的治疗效果良好。图6B为大鼠治疗前后评分变化，模型组在持续的OVA刺激后，评分大幅上涨，且大鼠死亡率持续升高(模型时死亡率为2/54＝3.7％，后期给药治疗期的死亡率为2/8＝25％)；合成肽sp2中剂量组和高剂量组在治疗后，评分也是上涨，提示sp2并未完全抵消OVA刺激的负面作用；合成肽brap高剂量、中剂量和低剂量组在治疗后，评分下降，提示合成肽brap完全抵消治疗期间的OVA刺激作用，且对大鼠恢复正常有一定的效果。
实施例6合成肽brap鼻腔给药对缓激肽诱发豚鼠肺微血管渗漏的影响
本技术由上海美轩生物科技有限公司提供。
一.动物分组及处理
雄性豚鼠体重为400g±5％，随机分为6组，6只/每组。
1.正常对照组：足外侧静脉注射等体积的生理盐水连续3天；
2.模型对照组：足外侧静脉注射等体积的生理盐水连续3天；
3.阳性对照组：足外侧静脉注射，地塞米松1.25mg/3ml/kg BW连续3天；
4.合成肽brap高剂量组：用微量加样枪给豚鼠双侧鼻腔注入浓度为3.0mM/L的合成肽brap，50μl/每侧/每天，连续3天；
5.合成肽brap中剂量组：用微量加样枪给豚鼠双侧鼻腔注入浓度为1.0mM/L的合成肽brap，50μl/每侧/每天，连续3天；
6.合成肽brap低剂量组：用微量加样枪给豚鼠双侧鼻腔注入浓度为0.33mM/L的合成肽brap，50μl/每侧/每天，连续3天。
二、豚鼠肺渗漏检测
1.连续给药3天，末次给药后20min，足外侧静脉相继注射l％EB(20mg/kg)和缓激肽15nmoL/kg(用生理盐水稀释为1 0nmoL/mL，按1.5mL/kg体重即15nmoL/kg)；正常组不注射EB和缓激肽。
2.肺组织EB测定：注射缓激肽30min后，将动物麻醉后颈动脉取血处死，做血常规，打开胸腔，剪开右心室和左心房，肺动脉插管，以30mL生理盐水灌洗肺循环，至流出液澄清为止.取出右肺下叶，生理盐水冲洗表面血液，滤纸吸干水分；取100mg剪碎，置于2mL甲酰胺溶液中，45℃水浴箱中孵育24h。1500rpm/min，离心5min，取上清，用620nm波长测定吸光度.由EB标准曲线(EB标准曲线如图7A所示)求出肺组织EB含量，如图7B所示。
3.EB标准曲线的制备：精确取0.1％EB溶液0.1mL，加入0.9mL生理盐水配制成0.0l％EB标准液lmL，取0.1mLEB标准液加甲酰胺至2mL，终浓度为5mg/L，用甲酰胺稀释为4、2、1、0.5mg/L。在620nm波长处测定吸光度OD值，OD值为纵坐标，EB浓度为横坐标，绘制OD值—EB浓度标准曲线，如图7A所示。
三、实验分析
1、伊文思蓝(EB)可与白蛋白结合，EB的渗出反应蛋白质的渗出情况，通过测定肺组织中渗漏的EB的含量，反应肺微血管通透性、肺微血管渗漏程度。与正常对照组比较，模型组肺组织EB含量显著增加(**表示，P<0.01)；合成肽brap低、中、高剂量组均明显低于模型组(P<0.01)，具有剂量-反应关系。与阳性对照组比较除低剂量组外无明显差异(^^表示，P>0.05)；
2、与正常对照组比较，模型组肺组织EB含量显著增加(**表示，P<0.001)；合成肽brap低、中、高剂量组均明显低于模型组(P<0.01)，具有剂量-反应关系。与阳性对照组比较无明显差异(^^表示，P>0.05)。
实施例7 Bradykinin B1受体靶点与brap的对接及结合模式
本技术由南京欧际医药科技服务有限公司提供。
1.采用NCBI Blast选取Bradykinin(BK)的B1 receptor(B1R)模板，基于PDB库的序列比对，选取了序列相似性和覆盖百分比较好的5UNF为模板。
2.采用Schrodinger软件包中的同源模建模块Prime对B1进行了三维结构模建。
根据模建的参数设置不同获得了两种B1的结构文件，分别是基于知识的受体结构和基于能量的受体结构。
3.采用MDockPeP将合成肽brap对接到两种受体结构上，总共进行了2次对接实验。
4.将对接实验中的最佳结合模式进行了brap-受体结合相互作用的能量分解，揭示了合成肽brap中氨基酸残基的能量贡献和与B1受体的结合模式。
①Bradykinin(BK)B1受体结构的同源模建
B1受体的序列从uniprot库中下载，获得同源模建中采用的B1受体的序列如SEQ ID No.7所示。
②受体Bradykinin(BK)B1 receptor与合成肽brap的分子对接:
采用MDockPeP进行B1受体蛋白和brap的结构的对接。
brap是由8种氨基酸组成的10肽化合物。
图中绿色代表了受体蛋白分子，浅蓝色代表了合成肽brap分子。
③计算Model中合成肽brap上每一个残基的能量贡献，
进一步研究B1(受体)与brap(配体)的相互作用残基，对于brap上的每一个残基，定义距离小于
Figure PCTCN2020100629-appb-000003
范围内的受体残基为接触残基，以此为基础，计算了brap上每一个残基的能量贡献
图中绿色代表了受体蛋白分子，浅蓝色代表了brap分子。
合成肽brap以U型结合于受体口袋中。
实施例8合成肽brap对缓激肽B1受体的抑制作用
本技术由武汉合研生物医药科技有限公司提供。
1.HEK293/G15/Bradykinin1实验方法:
复苏细胞:
将需要复苏的HEK293/Gα15/B1细胞从液氮罐内迅速取出，37℃水浴中融化。迅速将细胞悬液加入预热的DMEM+10％FBS培养基中，放入离心机，1000转/分钟，离心5分钟。将离心管取出，弃去上清液，向离心管内加入新鲜预热的培养基，重悬细胞，将细胞悬液加入培养皿，37℃，5％CO 2培养。
细胞传代:
稳定表达B1受体的HEK293/Gα15/B1细胞培养于DMEM+10％FBS；细胞培养条件：HEK293/Gα15/B1细胞系常规培养，含有10％胎牛血清和DMEM中传代。
当细胞长满培养皿80～90％，0.25％胰酶消化细胞，用新的培养基将细胞重悬，将细胞按适当比例传代，约2～3d传代1次。
实验过程:
1.将生长汇合度达到80％的HEK293/G15/Bradykinin1细胞用胰酶消化后计数，按照每孔2x10 4个/mL的密度铺于提前包被过matrigel的384孔黑边透明的384孔细胞培养板中。
2.将铺好的384孔细胞培养板放入5％CO 2 37℃培养箱中过夜培养。
3.实验当天将化合物合成肽brap用HBSS溶解成30mM的储备液。
4. 384孔板每孔加入10uL 4X免洗Fluo8染料，室温孵育1小时。
5.细胞在孵育的过程中，按照五倍稀释使用含有0.1％BSA的HBSS稀释待测化合物。阳性抑制剂起始浓度为10uM。
6.EC10的刺激终浓度为0.3nM，EC20刺激剂终浓度为0.6nM，EC80刺激剂终浓度为18nM。
7.将配好的化合物加到细胞培养板中,然后在FLIPR中，进行数据记录。
实验结果如图9所示。brap和Vehicle,EC10和EC20的Bradykinin混合溶液加到细胞体系中后，继续孵育15分钟后，加入EC80的刺激剂测试化合物抑制活性。
结果显示brap和EC80共同作用S型曲线明显，表明对B1受体的过度激活有明显抑制作用；与EC10和EC20共同作用的化合物效果不明显，推测是前一步反应已经释放了太多的钙离子，无法短期引起二次信号。
实施例9合成肽brap在LPS诱发的小鼠急性肺损伤中的保护作用
本技术由上海美轩生物科技有限公司提供。
一、动物分组与处理
1.SPF级别的Balb/C雄性小鼠，体重20±2g，n＝48
2.饲养条件：SPF无菌环境饲养，25度恒温环境，12h交替光照，充足食物水供给。
3.分组与处理：随机分成8组(A-H)、6只/每组；
3-1.造模前分别给予连续3天不同处理：
A正常对照组：尾静脉注射生理盐水1次/天；
B.LPS模型组：尾静脉注射生理盐水1次/天；
C.模型给药sp2组：尾静脉注射sp2(16mg/kgBW)1次/天；
D.模型给药brap高剂量组:尾静脉注射brap(16mg/kgBW)1次/天；
E.模型给药brap中剂量组:尾静脉注射brap(8mg/kgBW)1次/天；
F.模型给药brap低剂量组:静脉注射brap(4mg/kgBW)1次/天；
G.模型给药brap鼻腔给药组组：brap(3mM)，两侧鼻腔给药，15ul/每侧；1次/天；
H.模型给地塞米松组(阳性对照组)：地塞米松dexamethasome，DEX,5mg/kg腹腔注射，1次/天。
3-2.实验当日,即造模当日：
A-H各组动物继续分别给予上述处理30min后,A组给予等积生理盐水腹腔注射；其它各组均在30min后，给LPS 5mg/kgBW腹腔注射。
二、肺组织HE染色病理检测
实验步骤
1、组织切片、展片；
2、组织脱蜡水化；
3、苏木素染液染色5-20min(根据不同组织和实验要求调整)，自来水冲洗；
4、分化液分化30s；
5、自来水浸泡15min或温水(约50度)5min；
6、置伊红染液2min(根据不同组织和实验要求调整)，自来水冲洗；
7、自来水浸泡5min；
8、梯度酒精脱水：95％、100％I、100％II各1min；
9、二甲苯透明：二甲苯I、II各10min；
10、中性树胶封片；
11、放入60度烘箱中烤干，显微镜下观察。
实验结果如图16所示。
结果分析:
模型组：肺间隔增厚、炎细胞浸润、肺间隔有局灶性相互融合。brap静脉给药高剂量组(D)和brap鼻腔给药组(G)均未异常。
图16B为LPS模型组与正常对照组图16A比较，肺泡间隔增厚、炎细胞浸润、肺间隔有局灶性相互融合；图16C为LPS+合成肽sp2组，炎细胞浸润明显；图16D为LPS+brap高剂量组，未见肺泡间隔增厚、未见明显炎细胞浸润；E和F组分别为LPS+brap的中、低剂量组，随brap剂量降低，开始出现炎性细胞浸润；G组为鼻腔给药组，与D组相似，未见明显异常；H组为地塞米松阳性对照组,炎细胞浸润较明显，未见肺间隔局灶性相互融合。
实施例10血样本内毒素含量检测
血样本内毒素含量检测
1仪器
仪器 厂家 货号
多功能酶标仪 北京普朗 DNM-9602
2试剂
试剂 厂家 货号
内毒素检测鲎试剂盒 厦门鲎试剂生物科技有限公司 EC80545
实验步骤:
按内毒素检测鲎试剂盒说明书操作:
(1)取实施例9构建的各组小鼠的抗凝血，3000rpm/min离心2min，取100ul上清液，加入0.9ml样本处理液；
(2)将上述样本放入70℃干热仪加热10min后，用流水冷却；
(3)内毒素标准品的浓度梯度为1.0、0.5、0.25和0.1EU/ml；
(4)取数支无内毒素污染试管，分别加入100ul细菌内毒素检测用水、内毒素标准品和受试品；
(6)加入100ul鲎试剂溶液，混匀，盖上锡箔纸，37℃温育10min；
(7)加入100ul显色基质溶液，混匀，37℃温育6min；
(8)加入500ul偶氮化试剂1溶液，混匀；
(9)加入500ul偶氮化试剂2溶液，混匀；
(10)加入500ul偶氮化试剂3溶液，混匀，静置5min；
(11)545nm处测定吸光度值。
实验结果如图10所示。与正常组相比，LPS 5mg/kgBW腹腔注射后6h，小鼠血液内毒素水平显著提高，差异具有显著性；和模型组相比，合成肽brap低、中、高剂量组血液内毒素水平均明显低于模型组，具有剂量-反应关系。
实施例11 qPCR检测肺组织IL6 mRNA表达
实验步骤
试剂
试剂名称 厂家 货号
磁珠法总RNA提取试剂盒 上海美轩生物科技有限公司 MX0015
RT reagent Kit Takara RR047A
SYBRPremix Ex Taq 上海美轩生物科技有限公司 MX200017
Nuclease-free water Ambion cat#AM99386
乙醇 国药集团化学试剂有限公司 分析纯AR10009218
三氯甲烷 上海试剂一厂 试2006-06-08
1.样本准备
1.1肺组织样本：取实施例9构建的各组小鼠的新鲜肺组织样本，尽快用液氮速冻，-80℃保存。
2.总RNA的提取(采用磁珠法总RNA提取试剂盒)
2.1将组织样本切成小块后，液氮研磨(50mg)成粉状，转移到不含RNA酶的1.5ml管子中(细胞样本及其它液体样本无需研磨，直接跳转下一步)。
2.2总RNA提取
3.qPCR反应
应用Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System和 StepOnePlusTMReal-Time PCR System的操作方法
1.配制PCR反应液
试剂使用量
Figure PCTCN2020100629-appb-000004
Ex Taq   10.0μl
酶混合物                2.5μl
PCR Forward Primer(10μM)1μl
PCR Reverse Primer(10μM)1μl
PCR反转录Primer(10uM)0.5μl
RNA模板2.0μl
dH 2O(灭菌蒸馏水)3.0μl
Total 20.0μl
2.进行Real Time PCR反应。
3.实验结果分析。
反应结束后确认Real Time PCR的扩增曲线和融解曲线，计算2 -△△ct值等。
4.引物序列
Figure PCTCN2020100629-appb-000005
实验结果如图11所示，结果表明，合成肽brap静脉注射或鼻腔给予均明显降低LPS诱发的小鼠肺组织IL-6 mRNA的过表达。
实施例12血清细胞因子含量检测
试剂
试剂 厂家 货号
Mouse TNFa elisa kit MEXN M0047
Mouse IL6 elisa kit MEXN M0042
实验步骤
1、将试剂盒内试剂取出，常温平衡30min；
2、设置标准品孔和样本孔，标准品各孔分别加入不同浓度的标准品50ul；
3、待测样本孔先加入50ul待测样本(实施例9构建的各组小鼠模型的血液样本)；
4、标准孔和待测孔每孔各加入100ul HRP标记检测抗体；
5、封板膜封住反应孔，37℃孵育60min；
6、弃去液体，吸水纸上拍干，每孔加满洗涤液，静置1min；
7、弃去培养液，吸水纸上拍干，重复操作5次步骤2～步骤6；
8、每孔加入底物A\B各50ul，37℃孵育15min；
9、每孔加入终止液50ul，15min内酶标仪检测450纳米的OD值。
实验结果如图12、13所示：
1.和正常组相比，LPS(5mg/kgBW腹腔注射)诱发小鼠血清TNF-a和IL-6水平显著提高，差异具有显著性(**表示，p值＜0.01)；
2.和模型组相比，静脉注射brap低、中、高剂量组血清TNF-a和IL-6水平均明显降低具有剂量-反应关系(^^表示，p值＜0.01)；brap滴鼻组可以降低LPS,12h血液TNF-a和IL-6水平，P＜0.05。
实施例13流式细胞仪检测各组肺组织中ROS的荧光强度
实验步骤
1、取实施例9构架的各组小鼠模型的新鲜左肺组织100mg左右，PBS清洗三次去除血液；
2、将组织放入尼龙网中，用研磨棒研磨，同时用PBS冲洗；
3、收集细胞悬液，用200目筛网过滤去除组织块；
4、收集细胞悬液，1000rpm/min离心5min；
5、去除细胞上清，加入PBS重悬；
6、用无血清培养液配置10umol/L的DCFH-DA；
7、细胞离心去除PBS，加入上述探针稀释液，使细胞浓度为10 6个/ml；
8、37℃孵育30min，每隔3min颠掉一次；
9、1000rpm/min离心5min，用无血清培养基重悬细胞沉淀；
10、重复步骤9二次；
11、加入500ul PBS重悬细胞，流式细胞仪检测平均荧光强度值。
实验结果如图14所示，图14A为正常对照组，图14B为LPS模型组(5mg/kgBW腹腔注射)，图14C为LPS+16mg/kgBW合成肽brap，图14D为LPS+8mg/kgBW合成肽brap，图14E为LPS+4mg/kgBW合成肽brap，图14F为LPS+合成肽brap(两侧鼻腔注射，15μL/每侧/天)，图14G为阳性对照组(LPS+地塞米松5mg/kg腹腔注射)。
图15为流式细胞仪检测各组小鼠肺组织中ROS的平均荧光强度。
实施例14 brap静脉注射2000mg/kgBW剂量水平的限度实验
实验条件:Good Laboratory Practice，GLP
一.受试动物:昆明种小鼠健康、成年、雄性，体重≤20g，n＝5只；
二.受试样品:合成肽brap
1.Lot No.:04010039572，由苏卅强耀生物科技有限公司合成
2.含量:样品为精确分装，不用再称量，5mg/瓶x 8瓶(供第1只小鼠用)、8mg/瓶x 20瓶(供另外4只小鼠用)；
3.保存条件:保存-20 0C
4.样品纯度:HPLC检测纯度>99.49％
5.配制方法:从-20度取出合成肽brap冻干粉，室温恢复放置15min，加入1.5ml生理盐水，震荡，完全溶解。
三.给药途径及剂量:尾静脉注射2000mg/kgBW，
四.给药方式：
给药容量:小鼠尾静脉注射容量用最大容量即0.5ml/每次，缓慢注射
给药剂量和方式:40mg/1.5ml的工作液，在9h内，分3次
五.实验步骤:
1)将剂量为2000mg/kg的受试物(合成肽brap)给第1只昆明种小鼠进行尾静脉注射:
2)观察并记录是否出现毒性反应(起始症状、起始时间即给药后时间、严重程度及持续時间)
3)如果动物死亡，记录濒临死亡前的反应、死亡出现时间。
表.2.合成肽brap静脉注射2000mg/kg BW剂量水平的限度实验
Figure PCTCN2020100629-appb-000006
结果判断：如果存活的动物数≥3只，LD50大于2000mg/kg。
结论:静脉注射2000mg/kgBW剂量的限度实验未见毒性。

Claims (3)

  1. 一种合成肽brap，其特征在于，所述合成肽brap的氨基酸序列如SEQ ID No.1所示。
  2. 根据权利要求1所述的合成肽brap的如下(1)～(4)中任一所述的应用：(1)制备抑制G蛋白偶联的缓激肽B1和B2受体药物；(2)制备抗急性肺损伤药物；(3)制备新冠肺炎抗炎药物；(4)制备抗过敏性鼻炎药物。
  3. 根据权利要求2所述的应用，其特征在于，(1)～(4)中任一所述的药物的给药方式为静脉给药或鼻腔给药。
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