Coral microorganism-derived pro-angiogenic peptide cAMP-137 and application thereof

Abstract

The present application relates to the field of marine technology, and particularly relates to a coral microbial source pro-angiogenic peptide cAMP-137 and application thereof.In the present application, a novel pro-angiogenic peptide cAMP-137 is screened and identified for the first time from the metagenomic sequencing data of a sample of Pacific island coral reef.The pro-angiogenic peptide can significantly inhibit hematoma expansion and stabilize cerebral microvessels in a treatment window of 10-100 nM, filling the technical blank of existing polypeptide drugs and providing a new solution for spontaneous and drug-induced cerebral hemorrhage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of marine technology, in particular to a coral microbial-derived pro-angiogenic peptide cAMP-137 and its application. BACKGROUND

[0002] Spontaneous intracerebral hemorrhage (ICH) is the most devastating subtype of stroke, accounting for 10-15% of global stroke cases. Its condition is dangerous, with a 30-day mortality rate of 35-52%, and more than half of the survivors will leave severe neurological dysfunction. Etiologically, hypertensive cerebral arteriopathy is the main pathogenic factor of ICH, followed by cerebral amyloid angiopathy, vascular malformation and hemorrhage related to the use of anti-platelet or anticoagulant drugs.

[0003] At present, the clinical treatment strategy of ICH mainly focuses on acute phase life support and complication management, and there is a lack of specific drugs that can fundamentally improve the prognosis. Existing interventions can be divided into the following categories, but all have significant shortcomings:

[0004] Acute hemostatic drugs: such as antifibrinolytic drug tranexamic acid and pro-coagulant drug recombinant activated factor VIIa. They can limit the inhibition of hematoma expansion when applied in the super-early stage (within 1-2 hours after onset), but the treatment time window is extremely narrow, and is accompanied by an increased risk of thrombotic events (such as rFVIIa) or epilepsy, etc. The clinical benefit is limited, and the guidelines only recommend it as an auxiliary means for selective cases.

[0005] Anticoagulant reversal agents: for oral anticoagulant-related ICH, although they can quickly reverse coagulation function (such as 4-factor prothrombin complex, idarucizumab, etc.), but the drug efficacy maintenance time is short, there may be an "anti-coagulation activity rebound", and the high cost of the drug and strict storage requirements limit its popularization and application in primary hospitals.

[0006] Hypotensive and intracranial pressure-lowering drugs: intravenous hypotensive drugs (such as nicardipine) can control the risk of hematoma expansion, but the hypotensive process needs to be precisely controlled to prevent cerebral perfusion deficiency; osmotic diuretics (such as mannitol) can reduce intracranial pressure in the short term, but long-term use can easily lead to electrolyte imbalance, renal dysfunction and rebound intracranial hypertension.

[0007] Neuroprotective agents: such as edaravone and fentanyl, whose mechanism of action is mainly aimed at secondary injury (such as oxidative stress), however, large-scale clinical studies have failed to consistently demonstrate that they can significantly improve long-term neurological outcomes, and the frequent dosing regimen increases the treatment burden.

[0008] Prophylactic drugs: including low-dose anticoagulants for the prevention of deep vein thrombosis and antihypertensive and lipid-lowering drugs for long-term secondary prevention. The former has the problem of choosing the timing of re-administration after bleeding stabilizes and the risk of bleeding; the latter has poor patient compliance due to the complexity of the multi-drug combination regimen, and high-dose statins are associated with an increased risk of bleeding in some patients, and there is a lack of uniform standards for clinical decision-making.

[0009] In addition, it is worth noting that statins (HMG-CoA reductase inhibitors) widely used in the primary and secondary prevention of cardiovascular and cerebrovascular diseases have been suggested in several clinical studies (such as the SPARCL trial) that high-dose application may increase the risk of ICH. The potential mechanisms include: (1) inducing endothelial cell apoptosis and abnormal VEGF signaling, damaging the integrity of the microvascular wall; (2) down-regulating cholesterol, weakening the structural stability of the vascular wall; (3) exhibiting a dose-dependent biphasic effect - low doses can promote angiogenesis, while high doses lead to impaired blood-brain barrier function. This phenomenon reveals that drug-induced vascular fragility is an important mechanism for the occurrence and development of ICH, providing a new intervention target for the prevention and treatment of ICH.

[0010] In the field of drug development, some existing technologies have explored the use of polypeptide or protein drugs for ICH treatment. For example, a fusion peptide targeting IL-6R is used to inhibit neuroinflammation, a variant Factor Xa is used to reverse anticoagulant-related bleeding, and a lactoferrin-Fc fusion peptide is used to reduce iron toxicity. However, these existing technologies have common limitations: (1) single target: most of them only target a secondary link in the pathological process of ICH (such as inflammation or coagulation), and fail to simultaneously address the three core needs of rapid hemostasis, stable blood vessels to prevent hematoma expansion, and protection of neural units; (2) narrow therapeutic window: the effective concentration is close to the toxic concentration, lacking a wide range of safe concentration, limiting the reliability of its clinical application; (3) delivery obstacles: the in vivo stability, targeting, and bioavailability of polypeptide drugs restrict their clinical translation. Therefore, existing technologies have not yet disclosed an ICH treatment drug that can simultaneously achieve the synergistic effects of rapid hemostasis, vascular stability, and neural protection at low nanomolar concentrations. Therefore, the development of an innovative drug that can simultaneously and synergistically act on hemostasis, vascular stability, and neural protection in a wide therapeutic window, especially targeting the new target of "vascular fragility" revealed by statins, has become a key to filling the gaps in existing technologies and meeting the urgent needs of clinical practice. SUMMARY

[0011] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application aims to provide a coral microorganism-derived pro-angiogenic peptide cAMP-137 and its application. In the present application, a novel pro-angiogenic peptide cAMP-137 is screened and identified for the first time from the metagenomic sequencing data of a Pacific island coral reef sample. The pro-angiogenic peptide can significantly inhibit hematoma expansion and stabilize cerebral microvessels within a therapeutic window of 10-100 nM, filling the technical gap of existing polypeptide drugs and providing a new solution for spontaneous and drug-induced cerebral hemorrhage.

[0012] In a first aspect of the present application, a peptide or a functional fragment thereof is provided, wherein the peptide or the functional fragment thereof comprises:

[0013] (1) an amino acid sequence as shown in SEQ ID NO: 1; or

[0014] (2) a variant obtained by addition, deletion or substitution of at least one amino acid residue based on SEQ ID NO: 1, wherein the variant retains the same function as SEQ ID NO: 1.

[0015] In the present application, the term "functional fragment" refers to the smallest sequence fragment that can maintain at least one function of the pro-angiogenic peptide. "Maintaining function" means not losing the function, including: improving or reducing the function, but not completely losing the function. Generally, the maintenance of such function is based on conservative amino acid substitution (such as amino acid substitution achieved by conventional substitution means, such as alanine scanning). In the present application, "maintaining function" includes maintaining at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the original function (or activity). In the present application, the function includes pro-angiogenic and / or treatment of ICH.

[0016] In some embodiments of the present application, the peptide or the functional fragment thereof is an amino acid sequence as shown in SEQ ID NO: 1.

[0017] In some embodiments of the present application, the peptide or the functional fragment thereof is a single-chain linear structure, in the form of white powder, and is difficult to dissolve in water.

[0018] In some embodiments of the present application, the variant has at least 90% sequence identity with SEQ ID NO: 1.

[0019] In the present application, the term "sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences, and introducing gaps, if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment for purposes of determining percent amino acid sequence identity can be achieved by using various methods known to one of skill in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR). Those skilled in the art will appreciate that there are a variety of algorithms known in the art that can be used to determine the percent sequence identity between two sequences.

[0020] In some embodiments of the present application, the sequence identity can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0021] In some embodiments of the present application, the variant is a variant obtained by adding at least one amino acid residue to SEQ ID NO: 1.

[0022] In some embodiments of the present application, the variant comprises an amino acid sequence obtained by further adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues to SEQ ID NO: 1.

[0023] In some embodiments of the present application, the variant still contains the amino acid sequence shown in SEQ ID NO: 1.

[0024] In the present application, the terms "peptide", "polypeptide", "variant", "peptide fragment", "protein", or "polypeptide" are used interchangeably and refer to polymers of amino acids of any length. The polymers can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass polypeptides that have been modified by natural processes, such as post-translational processes, as well as those that have been artificially modified, for example, by the covalent attachment of a synthetic compound. This definition also includes polypeptides containing one or more analogs of an amino acid residue that has been modified naturally or by intervention. Examples of amino acid analogs include but are not limited to unnatural amino acids, as well as other modifications known in the art.

[0025] In some embodiments of the present application, a functional element is added to or a chemical modification is made to the peptide or functional fragment thereof.

[0026] In some embodiments of the present application, the functional element comprises a modifying element and a reinforcing element.

[0027] In some embodiments of the present application, the modifying element comprises a tag sequence, a targeting peptide, a dye, biotin, an affinity ligand.

[0028] In some embodiments of the present application, the modifying element comprises but is not limited to a His-tag, a GST-tag, a maltose binding protein tag, a Strep-tag, a c-Myc tag, a HA tag, a FLAG tag, a V5 tag, an AviTag, a SNAP-tag, a SUMO tag.

[0029] In the present application, the "reinforcing element" refers to an element that improves at least one function of the pro-angiogenic peptide or functional fragment thereof, or, confers or improves the drugability, physiological distribution, bioavailability, selectivity or pharmacokinetic properties of the pro-angiogenic peptide or functional fragment thereof.

[0030] In some embodiments of the present application, the reinforcing element can improve at least one of the physiological distribution, bioavailability, selectivity or pharmacokinetic properties, including stability, solubility, cell membrane penetration, half-life, binding activity, bioavailability, cytotoxicity, clearance rate.

[0031] In some embodiments of the present application, the reinforcing element comprises but is not limited to a cyclic peptide structure, a fatty acid group, a cell-penetrating peptide, PEG, albumin, etc.

[0032] In some embodiments of the present application, the chemical modification comprises phosphorylation, acetylation, methylation, ubiquitination or ubiquitin-like modification, glycosylation, lipidation, cyclization, enzymatic cleavage, disulfide bond formation, hydroxylation.

[0033] In some embodiments of the present application, D-amino acids, N-methylation or head-to-tail cyclization can be introduced at sites where the peptide or functional fragment thereof is easily cleaved by peptidases to prolong the half-life and maintain its therapeutic window of 10-100 nM.

[0034] In some embodiments of the present application, the peptide or functional fragment thereof is derived from a marine animal or a marine microorganism.

[0035] In some embodiments of the present application, the marine microorganism comprises: Acanthopleuribacteraceae Variovorax.

[0036] In some embodiments of the present application, the peptide or functional fragment thereof can be obtained by any conventional method, including but not limited to solid-phase synthesis, biological extraction, biological synthesis, etc.

[0037] In some embodiments of the present application, the biological synthesis includes high-density fermentation production by a heterologous expression system.

[0038] In some embodiments of the present application, the peptide or functional fragment thereof is synthesized by solid-phase synthesis.

[0039] In some embodiments of the present application, the peptide or functional fragment thereof has a purity of ≥98% (confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS)).

[0040] In a second aspect of the present application, a product is provided, which comprises at least one of the following:

[0041] (1) the peptide or functional fragment thereof according to the above aspect;

[0042] (2) a nucleic acid molecule encoding the peptide or functional fragment thereof in (1);

[0043] (3) an expression vector containing the nucleic acid molecule in (2);

[0044] (4) a transformant containing the nucleic acid molecule in (2) and / or the expression vector in (3).

[0045] In some embodiments of the present application, the nucleic acid molecule in (2) is added with a functional element or is chemically modified.

[0046] In some embodiments of the present application, the nucleic acid molecule in (2) comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0047] In some embodiments of the present application, the functional element and chemical modification are as defined in the above aspect.

[0048] In some embodiments of the present application, the expression vector comprises a plasmid.

[0049] In the present application, the type of plasmid is not limited and any conventional plasmid vector in the art can be used.

[0050] In some embodiments of the present application, the transformant comprises a bacterium, fungus, virus, plant cell or animal cell.

[0051] In some embodiments of the present application, the plant cell or animal cell is not involved in propagation material.

[0052] In some embodiments of the application, the product further comprises: the live transformant, the inactivated transformant, the attenuated transformant, the culture of the transformant, the metabolite, the concentrated or dried product, or the composition containing them.

[0053] In some embodiments of the application, the culture comprises culture supernatant or fermentation product.

[0054] In some embodiments of the application, the product is provided in liquid form or solid form.

[0055] In some embodiments of the application, the product is in the form of a liquid, foam, cream, spray, powder (e.g. lyophilized powder), or gel.

[0056] In some embodiments of the application, the product is in the form of a powder, microencapsulated powder, capsule, tablet, lozenge, granule, oral liquid, suspension, emulsion, liquid preparation, sustained-release preparation, nano-preparation, or micro-encapsulated capsule.

[0057] In some embodiments of the application, the product further comprises one or more pharmaceutically acceptable carriers, food carriers, excipients, and / or adjuvants. The pharmaceutically acceptable adjuvants are well known to those skilled in the art.

[0058] In some embodiments of the application, the adjuvant can be selected from at least one of carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings.

[0059] In some embodiments of the application, the product comprises one or more of buffers, lyophilization protectants, preservatives, stabilizers, binders, compacting agents, lubricants, dispersion enhancers, disintegrants, antioxidants, flavorings, sweeteners, and colorants.

[0060] In some embodiments of the application, the product further comprises other active agents.

[0061] In some embodiments of the application, the other active agents include: drugs with pro-angiogenic activity or drugs for treating internal hemorrhage.

[0062] In a third aspect of the present application, a conjugated polypeptide or fusion protein is provided, which comprises the peptide or functional fragment thereof of the above-mentioned aspects, and at least one conjugate.

[0063] In some embodiments of the application, the conjugate comprises at least one of a drug, a carrier, or an excipient.

[0064] In some embodiments of the present application, the drug comprises a drug having pro-angiogenic activity or treating internal hemorrhage.

[0065] In some embodiments of the present application, the internal hemorrhage comprises at least one of cerebral hemorrhage, adrenal hemorrhage, and pancreatic hemorrhage.

[0066] In a fourth aspect of the present application, there is provided use of at least one of the peptide or functional fragment thereof, product, or conjugated polypeptide or fusion protein as described in the above aspects in the preparation of a pro-angiogenic drug.

[0067] In some embodiments of the present application, the pro-angiogenic drug can be used for preventing, improving, or treating diseases such as traumatic cerebral hemorrhage, spinal cord hemorrhage, and drug-induced hemorrhage.

[0068] In the present application, the peptide or functional fragment thereof can simultaneously reduce the hemorrhage area and hemorrhage signal intensity and improve the grading in an effective dose range, indicating that it belongs to the stable barrier and anti-permeation type hemostasis path. The technical means is homogeneous with the microvascular barrier destruction link shared by traumatic cerebral hemorrhage, spinal cord hemorrhage, and drug-induced (especially statin-related) hemorrhage, and therefore can also obtain consistent therapeutic effect, proving its feasibility in a wider cerebral / spinal cord hemorrhage scenario.

[0069] Specifically, after administration of the peptide or functional fragment thereof, the hemorrhage area and integrated optical density (IOD) decrease simultaneously, and the hemorrhage severity grading shifts from moderate / severe to mild / ultra-mild. Based on statistical analysis, it is found that there is a significant difference between the 10 nM and 100 nM concentration conditions compared with the model group. Among them, the hemorrhage area of the 100 nM treatment group decreases to 50% ± 5.2% of the model group, and presents an "anti-permeation-stable barrier" phenotype. Further, the inventors have found that the hemorrhage area and IOD are significantly positively correlated, proving that the consistent improvement of the two dimensions of indicators is due to the overall relief of the same pathological link. Although traumatic cerebral hemorrhage (especially the phenotype dominated by microvascular bleeding / microhemorrhage), spinal cord hemorrhage / contusion, and drug-induced central microvascular bleeding / microhemorrhage have certain differences in the location of hemorrhage, but overall, they are related to the pathological main shaft of central microvascular barrier (BBB / BSCB) permeability abnormality, endothelial tight junction destruction, and red blood cell extravasation. This main shaft is homogeneous with the barrier destruction-bleeding link induced by atorvastatin in the example, and cAMP-137 has shown the triple consistent technical effect of reducing the leakage "range" and "intensity" and improving the "severity grading" in this model. Therefore, even without pre-setting a single action mechanism, it can be determined that cAMP-137 can obtain consistent technical effect with the model in the aforementioned indications dominated by microvascular bleeding / barrier destruction.

[0070] In addition, the hemostatic pathway of cAMP-137 is to stabilize the microvascular barrier, reduce the paracellular permeability, and inhibit the extravasation of red blood cells, rather than using systemic pro-coagulation as a necessary means. This feature is consistent with the characteristics of the therapeutic window (10-100 nM effective, ineffective at both ends), suggesting that the pro-angiogenic peptide is a steady-state regulatory molecule, which is suitable for central hemorrhage scenarios (such as traumatic micro-hemorrhage, spinal cord micro-hemorrhage, and drug-induced bleeding) that are not suitable for excessive pro-coagulation, thereby providing a favorable balance between effectiveness and safety.

[0071] Moreover, in the present application, the zebrafish ICH model induced by atorvastatin is used to evaluate the therapeutic effect by adopting a three-read evaluation system of "bleeding area, IOD, and bleeding grading", which can correspond to the bleeding volume / leakage score, hemoglobin load / signal intensity, and severity grading in clinical or mammalian research, respectively, and belongs to the mapping of endpoints with homogeneous physical meaning. Therefore, the triple improvement achieved by cAMP-137 in the model can be extrapolated to the corresponding evaluation system of the aforementioned target indications.

[0072] The present application has the following beneficial effects:

[0073] The present application solves various toxic side effects of existing drugs, such as the thrombosis complication problem of small molecule anti-fibrinolytic and coagulation factor preparations, the dehydration and electrolyte disorder risk caused by hyperosmotic dehydration agents and long-term intravenous infusion, and the low potassium, low sodium, and kidney function damage accompanying acute mannitol hyperosmotic therapy, etc. At a concentration of 10-100 nM, the present application can specifically reduce the leakage of red blood cells, significantly reduce the brain hemorrhage area, and thus can be used for preparing a drug for preventing and / or treating intracerebral hemorrhage.

[0074] In the present application, the zebrafish embryo model is used as a disease verification model, effectively reducing the sample consumption, solving the problems of high cost of single drug screening and being not conducive to large-scale drug screening, and effectively saving the research and development cost. Moreover, the model can cover the mapping indicators with homogeneous physical meaning of the bleeding volume / leakage score, hemoglobin load / signal intensity, and severity grading in clinical or mammalian research, thereby achieving homogenized verification. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 Figure is the HPLC detection result graph of the pro-angiogenic peptide cAMP-137.

[0076] Figure 2 Figure is the LC-MS detection result graph of the pro-angiogenic peptide cAMP-137.

[0077] Figure 3 Figure is the microscope image of the zebrafish embryo brain hemorrhage after intervention of the pro-angiogenic peptide cAMP-137.

[0078] Figure 4 The multi-dimensional quantitative analysis chart of the pro-angiogenic peptide cAMP-137 regulating zebrafish cerebral hemorrhage, wherein A is a quantitative chart of the pro-angiogenic peptide cAMP-137 regulating the area of zebrafish cerebral hemorrhage; B is a quantitative chart of the pro-angiogenic peptide cAMP-137 regulating the integral optical density (IOD) of zebrafish cerebral hemorrhage; C is a fitting analysis chart of the correlation between the area of zebrafish cerebral hemorrhage and signal intensity; D is a regional quantitative distribution chart of the pathological grading of zebrafish cerebral hemorrhage; and E is a standardization division schematic diagram of the zebrafish cerebral hemorrhage detection area. DETAILED DESCRIPTION

[0079] The content of the present application is further described in detail through specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0080] Example 1

[0081] In this example, a method for synthesizing and purifying a coral-derived pro-angiogenic peptide is provided.

[0082] In this example, based on the previous research, the inventors found a coral-related peptide derived from the family of Sulfobugula by assembling and binning analysis of the metagenomic sequencing data of Pacific island coral reef samples. Based on the amino acid sequence, the peptide was chemically synthesized by solid-phase peptide synthesis, and the synthesized product was verified for purity and molecular weight by high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS), thereby serving as the pro-angiogenic peptide cAMP-137 in this example. Acanthopleuribacteraceae

[0083] Specifically, the amino acid sequence of the pro-angiogenic peptide cAMP-137 is VRRGLDCKITGCRRQ (SEQ ID NO: 1), and the corresponding nucleotide sequence is 5'-GTCCGGCGCGGATTGGATTGTAAAATCACGGGTTGCCGCCGCCAATGA-3' (SEQ ID NO: 2).

[0084] The Fmoc solid-phase synthesis technology was used to synthesize the polypeptide cAMP-137 with the amino acid sequence of SEQ ID NO: 1 from C to N on a solid-phase carrier resin.

[0085] Specifically, the solid-phase peptide synthesis (SPPS) steps of the pro-angiogenic peptide cAMP-137 are as follows:

[0086] ​(1) Using Fmoc solid-phase synthesis technology, the amino acid sequence is synthesized from C-terminal to N-terminal on the solid-phase carrier resin. First, the resin pretreatment and the coupling of the first amino acid and the capping are carried out: 0.2 mmol of resin is placed in a solid-phase reaction column or a synthesis reactor with a bottom filter membrane, washed with N,N-dimethylformamide (DMF) for 2 times, then dried, 1 mL of dichloromethane (DCM) is added, shaken for 1 min, then 0.5 mL of amino acid (AA) reagent and 0.5 mL of N,N-diisopropyl ethylamine (DIPEA) are added in turn, shaken for 30 min at room temperature, and the coupling of the first amino acid is completed. Then 0.5 mL of methanol and 0.5 mL of DIPEA are added to the reactor and the reaction is continued for 10 min, the filtrate is discarded, and the resin is washed with DMF for 3 times, and the filtrate is discarded, to obtain the peptide resin loaded with the first amino acid.

[0087] (2) Fmoc deprotection: 1.5 mL of DEAPA solution is added to the peptide resin obtained in the above step, so that the resin is completely immersed, shaken for 2 min at 45°C, and the filtrate is discarded. The deprotection reaction is repeated twice. Then the resin is washed with DMF for 4 times to obtain the free amino peptide resin with Fmoc protection group removed.

[0088] (3) Stepwise coupling of subsequent amino acids: after obtaining the free amino peptide resin according to step (2), the remaining Fmoc-protected amino acids are coupled in turn. Each amino acid is coupled twice without removing the Fmoc protection group. First coupling: 1.5 mL of AA reagent is added, then 0.5 mL of 1-hydroxybenzotriazole (HOBT) solution and 0.5 mL of N,N'-diisopropyl carbodiimide (DIC) solution are added, shaken for 20 min at 45°C, the filtrate is discarded, and the resin is washed with DMF for 4 times, then the filtrate is discarded; second coupling: 1.5 mL of AA reagent is added, 0.5 mL of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) solution and 0.5 mL of DIPEA solution are added, shaken for 20 min at 45°C, then the filtrate is discarded, the resin is washed with DMF for 4 times, and the filtrate is discarded, and the coupling of one amino acid is completed.

[0089] Steps (2) and (3) are repeated, and finally the full-length polypeptide SEQ ID NO: 1 loaded on the resin is obtained. After the coupling of the N-terminal amino acid and the completion of the last Fmoc deprotection, the resin is washed with DMF and DCM for 3 times respectively, and the resin is dried under reduced pressure for standby.

[0090] (4) Polypeptide cleavage and crude preparation: 5 mL cleavage solution was added to the dried resin for polypeptide cleavage and side chain deprotection. Specifically, the polypeptide was cleaved from the resin and the side chain protecting groups were removed by using a mixed cleavage solution containing trifluoroacetic acid (TFA) under slight shaking or magnetic stirring at room temperature for 2-3 h. The filtrate was collected by pressure filtration. Anhydrous methyl tert-butyl ether pre-cooled (4 °C) was slowly added to the filtrate to precipitate the pro-angiogenic peptide. Then, the supernatant was discarded and the precipitate was collected by centrifugation at 4000 rpm for 5 min. The precipitate was dissolved in 10 mL of a 50% ACN / water solution, pre-frozen at -80 °C, and then freeze-dried to obtain a crude powder of the pro-angiogenic peptide cAMP-137.

[0091] (5) HPLC purification and freeze-drying: The freeze-dried crude product was dissolved in a small amount of a water-acetonitrile mixed solvent, filtered through a 0.22 μm filter membrane after complete dissolution, and then a portion of the product was detected for the target molecular weight. The obtained product (i.e., the crude peptide cAMP-137) had a retention time of 11.914 min as detected by high performance liquid chromatography analysis.

[0092] The crude peptide acetonitrile aqueous solution was loaded onto a reverse phase high performance liquid chromatography (RP-HPLC) system for purification. A C18 reverse phase chromatographic column was selected, mobile phase A was purified water containing 0.1 vol% TFA, and mobile phase B was acetonitrile containing 0.1 vol% TFA. Gradient elution was used, and the volume fraction of mobile phase B was linearly increased from 5% to 45% within 0-50 min, and the detection wavelength was 220 nm. The elution fraction of the target peak around 33.6% ACN gradient was collected, and the pre-mixed sample of the collected fraction was detected by high performance liquid chromatography and mass spectrometry. After the detection was qualified, the fraction was combined, pre-frozen, and placed in a freeze-dryer to obtain freeze-dried purified pro-angiogenic peptide cAMP-137 white solid powder.

[0093] A small amount of freeze-dried and purified pro-angiogenic peptide cAMP-137 was taken and dissolved in an appropriate amount of buffer for analytical RP-HPLC verification.

[0094] In the verification analysis, a SHIMADZU shim-pack GIST chromatographic column (4.6 mm x 250 mm, 5 μm) was used, mobile phase A was deionized water containing 0.1 vol% trifluoroacetic acid (TFA), mobile phase B was acetonitrile containing 0.1 vol% TFA, speed 1.0 mL / min, detection wavelength 214 nm, and injection volume 20 μL.

[0095] Before injection, the sample (0.1 mg of pro-angiogenic peptide cAMP-137) was dissolved in a mixed solvent of 0.5 mL acetonitrile and water (volume ratio 1:9).

[0096] A gradient elution program was used (20% linear increase in mobile phase B to 80% over 0-20 min).

[0097] LC-MS detection conditions: sample preparation as for HPLC purification, nebulizer gas flow rate 1.50 mL / min, desolvation tube (CDL) temperature 250 °C, ion source module temperature 200 °C, ionization voltage (+4.5 kV), detector voltage -0.2 kV; mobile phase: equal volume ratio (50%:50%) of water and acetonitrile mixture, flow rate 0.2 mL / min.

[0098] The results are shown in Figure 1 and Figure 2 .

[0099] HPLC analysis found that the purity of the synthesized pro-angiogenic peptide cAMP-137 was greater than 95%, and sequencing found that the sequence was correct. The theoretical molecular weight was 1761.13 Da, and mass spectrometry detection results ( Figure 2 ) showed that the molecular weight of the synthesized product was consistent with the theoretical value (error <0.01%), confirming the success of the synthesis.

[0100] The obtained pro-angiogenic peptide cAMP-137 was dissolved in 7% dimethyl sulfoxide (DMSO) to prepare a 1 mM pro-angiogenic peptide cAMP-137 stock solution, which was aliquoted into RNase / DNase-free cryotubes and stored for use.

[0101] Example 2

[0102] In this example, in order to verify the related efficacy of the above-mentioned pro-angiogenic peptide cAMP-137, an animal experiment was performed using an atorvastatin-induced zebrafish cerebral hemorrhage model as a disease animal model.

[0103] In this example, the zebrafish were strictly bred in accordance with the zebrafish breeding standards, i.e. the light cycle was set to 14 hours light / 10 hours dark, and the Daphnia magna larvae were fed at 8:00 am and 18:00 pm every day, with intermittent supplementation of conventional tropical fish feed. The breeding environment was maintained under conventional water quality conditions (pH 7.0-7.5, hardness 50-100 mg / L CaCO3.

[0104] AB strain zebrafish embryos were obtained by natural mating: healthy adult AB strain zebrafish (3-4 cm in length for females and 2.5-3.5 cm in length for males) were selected and placed in a mating tank at a ratio of 2:1. The next morning, the fertilized eggs were collected and placed in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.2-7.4). After washing three times, the embryos were transferred to a 28.5°C incubator.

[0105] At 4 hours post-fertilization (4 hpf), the embryos were screened under a stereomicroscope: dead embryos (no heartbeat and yolk sac movement), unfertilized embryos (opaque egg membranes without division), and morphologically abnormal embryos (yolk sac rupture, body axis distortion) were removed, and normal developing embryos were reserved for subsequent experiments.

[0106] The screened AB strain zebrafish embryos (24 hours post-fertilization (hpf)) were randomly divided into groups and transferred to sterile 24-well cell culture plates, with 15 embryos per well, and 1 mL of freshly prepared E3 medium (containing the corresponding test substance) was added. The specific grouping and test substance addition are shown below:

[0107] The blank control group: each well contained only 1 mL of E3 medium without any additives.

[0108] The model group: each well contained 1 mL of E3 medium containing atorvastatin at a final concentration of 0.5 μM.

[0109] The treatment group: each well contained 1 mL of E3 medium containing atorvastatin at a final concentration of 0.5 μM and gradient concentrations (1 nM, 10 nM, 100 nM, and 1 μM) of the pro-angiogenic peptide cAMP-137.

[0110] The 24-well plates were placed in a 28.5°C incubator for further incubation until 3 days post-fertilization (72 hpf), and then subjected to o-dianisidine staining. Specifically, zebrafish embryos cultured to 3 days post-fertilization (3 dpf) were fixed overnight at 4°C with 4% paraformaldehyde solution (containing 0.1 M PBS, pH 7.4). After washing three times with PBS buffer (5 minutes each time), the fixed embryos were transferred to a dark room environment and stained with 0.6 mg / mL o-dianisidine staining solution (0.01 M sodium acetate buffer (pH 4.5), 0.65 vol% hydrogen peroxide, and 40 vol% ethanol were mixed at a volume ratio of 5:1:4, and then o-dianisidine dye was added) for 15 minutes in the dark. After staining, the reaction was terminated with PBS buffer and observed under a stereomicroscope to collect images of the brain hemorrhagic area.

[0111] The brain hemorrhage area was quantitatively analyzed by using image analysis software. Specifically, the stained embryo image was imported, and the threshold parameter was set. Specifically, the stained embryo image was imported into the image analysis software. First, the image was uniformly adjusted to the same resolution and brightness, and converted to an 8-bit grayscale image. The threshold was set to 100-255 in the grayscale range of 0-255, the image was threshold segmented to obtain a binary image of the brain hemorrhage area, and the area of the hemorrhage area was measured based on this, and the integral optical density parameter of the area was recorded. Each sample was measured 3 times, and the average value was taken as the final result.

[0112] The results are shown in Figure 3 and Figure 4 .

[0113] In this embodiment, the improvement effect of pro-angiogenic peptide cAMP-137 on atorvastatin-induced zebrafish brain hemorrhage was systematically explored by multi-dimensional quantitative analysis. Specifically, in this embodiment, the brain hemorrhage area and integral optical density (IOD, reflecting the hemorrhage signal intensity) were taken as the core indicators. Through single factor variance analysis, it was found that the brain hemorrhage area and integral optical density of the 10 nM and 100 nM cAMP-137 treatment groups were significantly lower than those of the model group (atorvastatin treatment, * P<0.05, **** P<0.0001). Among them, the hemorrhage area of the 100 nM cAMP-137 treatment group was reduced to 50%±5.2% of the model group, thereby fully proving that the pro-angiogenic peptide cAMP-137 in this concentration range can effectively reduce the degree of brain hemorrhage. The brain hemorrhage area and integral optical density of the 1 nM and 1 μM cAMP-137 treatment groups had no statistical difference with the model group (ns), indicating that the anti-hemorrhage effect of the pro-angiogenic peptide cAMP-137 exists in a concentration window effect.

[0114] Further analysis combined with scatter plots found that the brain hemorrhage area and IOD were significantly positively correlated (the data points distribution embodied the synchronous change rule). Among them, the data points of the 10 nM (blue) and 100 nM (green) cAMP-137 treatment groups were concentrated in the low area-low IOD region, further illustrating that the improvement effect of the pro-angiogenic peptide cAMP-137 in this concentration range on brain hemorrhage was consistent, and was positively correlated with the dose.

[0115] The grading improvement of the severity of brain hemorrhage can be classified and quantified by four levels of ultra-mild, mild, moderate and severe. Among them, the quantification standard is: according to the zebrafish brain anatomy structure, the medulla oblongata, the midbrain and cerebellum, and the telencephalon are defined as hemorrhage area 1, area 2 and area 3, respectively. In the atorvastatin-induced brain hemorrhage model, whether each area has hemorrhage is used to grade and quantify the severity of brain hemorrhage:

[0116] Ultra-mild (0) is no hemorrhage in three brain regions;

[0117] Mild (1) is only 1 brain region with hemorrhage;

[0118] Moderate (2) is any 2 brain regions with hemorrhage;

[0119] Severe (3) is all three brain regions with hemorrhage.

[0120] From the above results, it can be seen that the model group is mainly moderate and severe hemorrhage (high proportion of dark sections), which confirms that atorvastatin can induce severe cerebral hemorrhage phenotype. In contrast, the proportion of ultra-mild and mild hemorrhage in the 10 and 100 nM cAMP-137 treatment groups increased significantly, and the proportion of severe hemorrhage decreased, indicating that the pro-angiogenic peptide cAMP-137 not only reduced the hemorrhage range, but also reduced the pathological severity of hemorrhage.

[0121] In order to ensure the spatial consistency of quantitative analysis, the inventors further standardized the positioning of zebrafish brain hemorrhage area (such as Figure 4 E in the specification), which is divided into the following 3 functional regions: region 1 (medulla oblongata), region 2 (midbrain / cerebellum / diencephalon), and region 3 (telencephalon), which can also better reflect the treatment effect of the pro-angiogenic peptide cAMP-137 in different regions, and also provides a standardized reference framework for corresponding positioning research itself.

[0122] In summary, it can be determined that the pro-angiogenic peptide cAMP-137 in the concentration range of 10 and 100 nM can improve atorvastatin-induced zebrafish cerebral hemorrhage by reducing the hemorrhage area, reducing the pathological grade, and synchronously inhibiting the signal intensity, and its effect has concentration specificity (invalid at both ends, effective in the middle).

[0123] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A pro-angiogenic peptide cAMP-137 derived from coral microorganisms, characterized in that, The amino acid sequence of the pro-angiogenic peptide cAMP-137 is shown in SEQ ID NO:

1.

2. A product, characterized in that, The product includes at least one of the following: (1) The angiogenesis peptide cAMP-137 according to claim 1; (2) The nucleic acid molecule encoding cAMP-137, the pro-angiogenic peptide in (1); (3) Expression vectors containing nucleic acid molecules from (2); (4) Transformants containing the nucleic acid molecules in (2) and / or the expression vector in (3).

3. The use of at least one of the angiogenic peptide cAMP-137 of claim 1 and the product of claim 2 in the preparation of a medicament for treating at least one of spinal cord hemorrhage and drug-induced hemorrhage.

4. The use of at least one of the angiogenic peptide cAMP-137 of claim 1 and the product of claim 2 in the preparation of a medicament for the prevention and / or treatment of at least one of cerebral hemorrhage, adrenal hemorrhage and pancreatic hemorrhage.

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