A cyclic peptide specifically recognizing complement C5a and preparation and use thereof

Abstract

The application discloses a cyclic peptide specifically recognizing complement C5a and a preparation and application thereof. The amino acid sequence of the cyclic peptide is shown as SEQ ID NO:1. The cyclic peptide has good plasma stability, and can be used for preventing and treating sepsis by adopting an intravenous injection administration mode and can significantly prolong the survival period of a CLP-induced sepsis model mouse.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a cyclic peptide that specifically recognizes complement C5a, its preparation and uses, and its use in the prevention and treatment of sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The physiological and pathological characteristics of sepsis are complex, and its disease progression is highly dynamic. Currently, most drugs used to treat sepsis have failed in clinical trials. Timely control of excessive inflammatory responses is crucial for the prevention and treatment of sepsis.

[0003] Complement activation is a crucial mechanism of inflammatory response in sepsis, leading to the release of potent anaphylatoxins (C3a and C5a) and membrane attack complexes (C5b-9). C5a, with its chemotactic capacity 20 times greater than C3a, plays a key role in complement cascade amplification. Excessive complement activation is critical to the clinical outcome of sepsis. Uncontrolled complement activation can exacerbate hyperinflammatory responses, activate coagulation cascades leading to disseminated intravascular coagulation (DIC), cause vascular leakage and tissue edema, and further worsen organ dysfunction. Simultaneously, excessive complement activation can impair neutrophil and macrophage function, leading to excessive neutrophil depletion, inducing thymocyte apoptosis, resulting in immunosuppression and increasing the risk of secondary infections. Early intervention to block excessive complement activation and effectively regulate the inflammatory response is of great significance for the prevention and treatment of sepsis. Therefore, targeted blocking of C5a can selectively block the C5a-C5aR axis, reducing excessive inflammatory response, while preserving important functions of the complement system, such as C3 / C3b-dependent opsonization and MAC-mediated bacterial lysis.

[0004] IFX-1 is an antibody drug targeting C5a developed overseas. It entered Phase II clinical trials for sepsis during the COVID-19 pandemic but has not yet received FDA approval. IFX-1 possesses highly effective blocking ability against C5a (K... D While the binding affinity of these antibodies is generally very high (pM level), the extremely high binding affinity significantly increases the risk of secondary infections. NOX-D20, another DNA / RNA hybrid aptamer drug targeting C5a developed abroad, also possesses a high binding affinity for C5a (0.1 nM), but it ultimately stopped at preclinical trials. Therefore, for the development of C5a-blocking drugs for the prevention and treatment of sepsis, suitable binding affinity is particularly important for inhibitors. At the same time, competitively low production costs are also crucial for expanding drug accessibility.

[0005] Plasma stability is a prerequisite for targeted drug delivery and efficacy. The widespread upregulation of plasma proteases in sepsis patients places even higher demands on the plasma stability of drugs used for sepsis prevention and treatment.

[0006] Currently, there are no reports of C5a-blocking cyclic peptide drugs used for the prevention and treatment of sepsis, either domestically or internationally. Summary of the Invention

[0007] This invention addresses the lack of C5a-blocking cyclic peptide drugs for the prevention and treatment of sepsis in the prior art, and provides a C5a-blocking cyclic peptide, the amino acid sequence of which is shown in SGHSMPMVFERF (Lactam Bridge: S1-F12, SEQ ID NO: 1). The cyclic peptide (Cp) provided by this invention specifically blocks the C5a-C5aR1 interaction without affecting the C5a-C5aR2 interaction, specifically binds to C5a without hindering C5a cleavage and the formation of the C5b-9 MAC membrane attack complex.

[0008] In one aspect, the present invention provides a cyclic peptide that specifically recognizes complement C5a, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] Another aspect of the present invention provides a modified polypeptide fragment comprising the amino acid sequence shown in SEQ ID NO:1, wherein the side chain protecting groups of Ser, His, Glu and Arg are tBu, Trt, OtBu and Pbf, respectively, and the side chain protecting groups of the two Ser in SEQ ID NO:1 are both tBu.

[0010] In some embodiments, the modified polypeptide fragment comprises the amino acid sequence shown in SEQ ID NO: 2.

[0011] S(tBu)GH(Trt)S(tBu)MPMVFE(OtBu)R(Pbf)F(SEQ ID NO: 2).

[0012] Another aspect of the present invention provides the application of the modified polypeptide fragment provided by the present invention in the preparation of a cyclic peptide that specifically recognizes complement C5a.

[0013] Another aspect of the present invention provides a method for synthesizing a cyclic peptide that specifically recognizes complement C5a, the method comprising the following steps:

[0014] (a) Using resin as a solid support, amino acid residues are coupled sequentially from the C-terminus to the N-terminus according to the sequence of SEQ ID NO:1, wherein the side chain protecting groups of Ser, His, Glu and Arg are tBu, Trt, OtBu and Pbf, respectively, and the side chain protecting groups of the two Ser in SEQ ID NO:1 are both tBu;

[0015] (b) After the linear peptide synthesis is completed, the straight-chain peptide is cleaved from the resin by lysis buffer A to obtain a straight-chain peptide with side chain protecting groups;

[0016] (c) Intramolecular cyclization of the product of step (b) in the presence of a condensation system; and,

[0017] (d) After cyclization, the side chain protecting groups were removed using lysis buffer B to obtain the cyclic peptide shown in SEQ ID NO: 1.

[0018] In this invention, lysis buffer A is different from lysis buffer B.

[0019] In some implementations, step (b) involves the lysis buffer A comprising TFA and DCM.

[0020] In some embodiments, the volume ratio of TFA to DCM in the lysis buffer A is (1-5):(95-99).

[0021] In some implementations, step (c) involves the condensation system comprising PyBop and DIEA.

[0022] In some implementations, step (d) involves the lysis buffer B comprising TFA, TIS, and H2O.

[0023] In some embodiments, the volume ratio of TFA:TIS:H2O in the lysis buffer B is (92-97):(2-3):(1-3).

[0024] In some implementations, step (c) cyclization is carried out at 0-5°C.

[0025] In some embodiments, the molar ratio of PyBop to amino acids or peptides in the condensation system is 1:(0.5-4).

[0026] In some embodiments, the molar ratio of PyBop to amino acids or peptides in the condensation system is 1:(1-4), for example 1:2.

[0027] In some embodiments, the molar ratio of DIEA to condensing agent in the condensation system is (0.5-2):(0.5-2).

[0028] In some embodiments, the molar ratio of DIEA to the condensing agent in the condensation system is 1:(0.5-2), for example 1:1.

[0029] In some implementations, the resin in step (a) is Fmoc-Phe-2-chlorotriphenylmethyl resin.

[0030] In some implementations, the cyclization reaction time in step (c) is 1-3 hours.

[0031] In some embodiments, the synthesis reaction further includes step (e), which involves RP-HPLC purification and lyophilization to obtain the cyclic peptide product.

[0032] In some embodiments, in step (e), the mobile phase is an aqueous or acetonitrile solution containing 0.05% trifluoroacetic acid.

[0033] In some implementations, the C-terminus of the cyclized propeptide is a carboxyl group and the N-terminus is an amino group.

[0034] In some implementations, amide bonds (-CO-NH-) are formed within the cyclic peptide molecule.

[0035] In some implementation schemes, the final purity of the cyclic peptide is ≥95% (HPLC detection).

[0036] In another aspect, the present invention provides a pharmaceutical composition comprising the cyclic peptide provided by the present invention.

[0037] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0038] In some embodiments, the pharmaceutically acceptable excipients include one or more of stabilizers, buffers, antioxidants, solubilizers, lyophilization protectants, and isotonic modifiers.

[0039] In some implementations, stabilizers (such as trehalose, sucrose, or arginine) prevent peptide chain aggregation; buffers (such as acetate, citrate, or histidine buffer, pH 4.0–7.0) maintain solution stability; antioxidants (such as methionine or EDTA) inhibit oxidative degradation; solubilizers (such as cyclodextrin or polysorbate 80) improve the solubility of hydrophobic cyclic peptides; lyophilization protectants (such as mannitol or glycine) are used for constructing the lyophilized formulation framework; and isotonic adjusters (such as sodium chloride or glycerol) ensure injection safety.

[0040] In some implementations, the cyclic peptide can be dissolved in histidine buffer (pH 6.0), with the addition of trehalose, methionine, and polysorbate 80, then lyophilized and reconstituted for injection.

[0041] In another aspect, the present invention provides a reagent or kit comprising the cyclic peptide or pharmaceutical composition provided by the present invention.

[0042] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for treating sepsis.

[0043] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for selectively blocking the C5a-C5aR1 signaling pathway without affecting the interaction of C5a-C5aR2.

[0044] Another aspect of the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for specifically binding C5a without inhibiting C5 cleavage and subsequent formation of the C5b-9 membrane attack complex.

[0045] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for alleviating excessive inflammatory response caused by excessive accumulation of C5a.

[0046] In this invention, "relief" refers to reducing, inhibiting or alleviating excessive or uncontrolled inflammatory responses caused by excessive accumulation of complement component C5a through intervention.

[0047] In some implementations, mitigating the excessive inflammatory response caused by excessive C5a accumulation is referred to as mitigating the excessive inflammatory response caused by sepsis-associated excessive C5a accumulation.

[0048] In some implementations, mitigating excessive inflammatory responses caused by excessive C5a accumulation involves inhibiting or reducing the levels of inflammatory factors in the plasma.

[0049] In some implementations, mitigating excessive inflammation caused by C5a over-accumulation involves inhibiting or reducing the levels of chemokines in the plasma.

[0050] In some implementations, the plasma is plasma from a sepsis model mouse.

[0051] In some implementations, the plasma is plasma from a sepsis patient.

[0052] In some embodiments, the inflammatory factors are selected from IL-6, TNF-α, and IL-1β.

[0053] In some implementations, the inflammatory factors are selected from IL-6 and TNF-α.

[0054] In some embodiments, the chemokine is selected from CXCL1, CXCL5, CCL2, CCL3, CCL4, CCL5, CCL7, and CCL12.

[0055] In some implementations, the chemokine is selected from CXCL1 and CCL2.

[0056] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for inhibiting C5a-induced chemotaxis of neutrophils.

[0057] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for prolonging the survival of a mouse model of sepsis.

[0058] In some embodiments, sepsis model mice using the cyclic peptide provided by the present invention have at least 2 or 3 times the survival time compared to sepsis model mice not using the cyclic peptide provided by the present invention.

[0059] In another aspect, the present invention provides the use of the cyclic peptide provided by the present invention in the preparation of a medicament for prolonging the survival of patients with sepsis.

[0060] In some embodiments, patients using the cyclic peptide provided by the present invention have a survival time that is at least 0.5 times longer than patients who do not use the cyclic peptide provided by the present invention.

[0061] The C5a blocking cyclic peptide in this invention can specifically recognize complement C5a in mice and humans, exhibiting excellent binding specificity and good plasma stability, and can be used for the effective prevention and treatment of sepsis.

[0062] the term:

[0063] C5a: A cleavage product of complement component C5, it is a potent inflammatory mediator that mediates inflammatory responses by binding to receptors C5aR1 and C5aR2.

[0064] C5a-C5aR1: The interaction between C5a and its receptor C5aR1 (C5a receptor 1) mainly promotes pro-inflammatory responses, cell chemotaxis, and immune cell activation.

[0065] C5a-C5aR2: The interaction between C5a and its receptor C5aR2 (C5a receptor 2) has a more complex regulatory function, involving anti-inflammatory or pro-inflammatory signaling pathways, and the specific function varies depending on the disease and cell type.

[0066] C5: A key component of the complement system, it cleaves to generate C5a and C5b, the latter of which participates in the formation of the membrane attack complex (MAC).

[0067] MAC (Membrane Attack Complex, C5b-9): A protein complex composed of complement components C5b, C6, C7, C8, and C9, which can form pores on the cell membranes of pathogens or abnormal cells, mediating cell lysis.

[0068] TFA (Trifluoroacetic Acid): Trifluoroacetic acid is commonly used as a solvent or additive in peptide synthesis and purification.

[0069] TCA (Trichloroacetic acid): Trichloroacetic acid is commonly used to precipitate proteins.

[0070] CLP (Cecal Ligation and Puncture): Cecal ligation and puncture, a widely used animal model of sepsis that simulates the pathological process of sepsis in humans.

[0071] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0072] The reagents and raw materials used in this invention are all commercially available.

[0073] The positive and progressive effects of this invention are as follows: the cyclic peptide (Cp) provided by this invention specifically blocks the interaction between C5a and C5aR1 without affecting the interaction between C5a and C5aR2, specifically binds to C5a without hindering C5 cleavage and the formation of the MAC membrane attack complex by C5b-9.

[0074] The cyclic peptide of this invention can effectively inhibit inflammatory factors and chemokines in the plasma of septic model mice; it can also effectively inhibit C5a-induced chemotaxis in mouse neutrophils.

[0075] The cyclic peptide of this invention has good plasma stability; when administered intravenously, it is used for the prevention and treatment of sepsis and can significantly prolong the survival of CLP-induced sepsis model mice. Attached Figure Description

[0076] Figure 1 The results show the binding affinity of the C5a-blocking cyclic peptide Cp to mouse complement C5a.

[0077] Figure 2 The results show the binding affinity of the C5a-blocking cyclic peptide Cp to human complement C5a.

[0078] Figure 3 The results are flow cytometry semi-quantitative results for the specific blocking of C5a-C5aR1 interaction by the C5a-blocking cyclic peptide Cp.

[0079] Figure 4The results of the hemolysis experiment show that C5a blocks the specific binding of the cyclic peptide Cp to C5a.

[0080] Figure 5 The results show the stability of the C5a-blocking cyclic peptide Cp in the plasma of CLP model mice.

[0081] Figure 6 ELISA results show the inhibitory effect of C5a-blocking cyclic peptide Cp on plasma inflammatory factor IL-6 in CLP-induced sepsis model mice.

[0082] Figure 7 ELISA results show the inhibitory effect of C5a-blocking cyclic peptide Cp on plasma inflammatory factor TNF-α in CLP-induced sepsis model mice.

[0083] Figure 8 ELISA results show the effect of C5a-blocking cyclic peptide Cp on the inhibition of plasma chemokine CXCL1 in CLP-induced sepsis model mice.

[0084] Figure 9 ELISA quantitative results of the inhibitory effect of C5a-blocking cyclic peptide Cp on plasma chemokine CCL2 in CLP-induced sepsis model mice.

[0085] Figure 10 This is a quantitative result of the inhibitory effect of the C5a-blocking cyclic peptide Cp on C5a-induced chemotaxis in mouse neutrophils.

[0086] Figure 11 The quantitative results show the inhibitory effect of the C5a-blocking cyclic peptide Cp on C5a-induced chemotaxis in human neutrophils.

[0087] Figure 12 The effect of C5a-blocking cyclic peptide Cp on prolonging the survival of CLP-induced sepsis model mice. Detailed Implementation

[0088] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0089] Sources and preparation of C5a-blocking cyclic peptide (Cp)

[0090] The Cp sequence of this invention was obtained by screening a 12-peptide library targeting mouse C5a protein (1-77) (UniProt ID: P06684) using phage display technology. Specifically, the phage display peptide library was screened by Camed Biotechnology.

[0091] The polypeptide Cp was obtained using a solid-phase synthesis method, as detailed below:

[0092] 1) Select 1g of Fmoc-Phe-2-ChlorotritylChloride Resin resin with a loading of 0.4 mmol / g, and remove the Fmoc protecting group after swelling with DCM;

[0093] 2) Weigh 777.6 mg of Fmoc-Arg(Pbf)-OH, 624.4 mg of PyBop (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate), and 0.52 mL of DIEA. Dissolve them in 10 mL of DMF at 0°C. After complete dissolution, add the solution to the reaction column and react at room temperature for 1 hour. The reaction progress is judged by a negative result using the ninhydrin detection method. After the coupling reaction is completed, the reaction solution is dried under vacuum and washed with 10 mL of DMF. Then, add 10 mL of 20% hexahydropyridine DMF solution and react for 5 min. Wash once with DMF (N,N-dimethylformamide). After washing, add 10 mL of 20% hexahydropyridine DMF solution again and react for 10 min. Drain the solution under vacuum and wash three times with DMF, twice with DCM, and once with DMF.

[0094] 3) According to the polypeptide sequence, coupling is performed sequentially from the C-terminus to the N-terminus until Fmoc-Ser(tbu)-OH and Fmoc is removed. The side chain protecting groups of Ser, His, Glu and Arg are tBu, Trt, OtBu and Pbf, respectively. All amino acids are protected with Fmoc at the α-amino position.

[0095] 4) After the coupling reaction is complete, the reaction solution is dried under vacuum, washed three times with 10 mL DMF, washed three times with methanol, dried, and the resin is weighed as 2.1 g.

[0096] 5) The peptide was reacted with a linear peptide resin using a lysis buffer (TFA:DCM = 2:98) to obtain approximately 825.6 mg of peptide with all side chain protecting groups. After preliminary separation and purification, the peptide was lyophilized.

[0097] 6) Add 416.2 mg of PyBop (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate) and 4.2 mL of DIEA to 5 mL of DMF at 0 °C. React for 2 h. After the reaction is complete, the filter cake after adding water and filtering is determined by mass spectrometry.

[0098] 7) The above filter cake was reacted with the lysis buffer (TFA:triisopropylsilane:water = 95:2.5:2.5) to obtain 576.2 mg of cyclic polypeptide with all side chain protecting groups removed, which was then dissolved in an appropriate amount of pure water.

[0099] 6) The crude linear peptide aqueous solution was filtered through a 0.45 μm filter membrane. The purified crude peptide was then purified using high-performance liquid chromatography (HPLC): a DAC-HB50 dynamic axial compression column was used with gradient elution using mobile phases A (0.05% trifluoroacetic acid aqueous solution) and B (0.05% trifluoroacetic acid acetonitrile solution). The sample was detected using a UV detector, and the peptide solution of the target peak was collected in fractions. After HPLC purification, 30 mL of a finished peptide liquid with a purity greater than 95% was obtained. This liquid was concentrated by rotary evaporation to obtain 5 mL of liquid. The liquid was then subjected to a salt transfer operation, pre-lyophilized, and lyophilized to finally obtain 5.5 mg of refined peptide.

[0100] Example 1: Determination of the binding affinity of C5a-blocking cyclic peptide (Cp)

[0101] The binding affinity of Cp (SGHSMPMVFERF (Lactam Bridge:S1-F12) (SEQ ID NO:1)) to mouse and human complement C5a was determined using a Biacore 2000 system (GE).

[0102] Surface plasmon resonance (SPR) analysis was performed using a Biacore 2000 system (GE Healthcare, Germany) at 37°C in a running buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2). The CM5 sensor chip (GE Healthcare) was activated using NHS / EDC, and mouse C5a (ACROBiosystems, China) and human C5a (ACROBiosystems, China) were then immobilized. Peptide-protein interactions were characterized using a concentration gradient method (0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM) at a continuous flow rate of 30 μL / min (injection time 150 s). Between analyte concentrations, the analyte was treated with 10 mM glycine-HCl (pH 2.0) for 5 min. The data were globally fitted to a 1:1 Langmuir binding model using Biacore Insight evaluation software (Cytiva, USA) to obtain binding and dissociation constants.

[0103] Figure 1 , Figure 2 The figures show the SPR (Split Reactivity Ratio) results of the C5a-blocking cyclic peptide Cp of this invention with mouse complement C5a and human complement C5a, respectively. Table 1 summarizes the dissociation constants (Kp) of the binding of the C5a-blocking cyclic peptide Cp of this invention with mouse complement C5a and human complement C5a. D ), binding rate constant (K)a ) and dissociation rate constant (K d ). Figure 1 , Figure 2 The SPR results showed that the C5a blocking cyclic peptide Cp of this invention has comparable binding affinity to mouse complement C5a and human complement C5a.

[0104] Table 1 shows the dissociation constants (Kp) of the C5a-blocking cyclic peptide Cp with mouse and human complement C5a. D ), binding rate constant (K) a ) and dissociation rate constant (K d Summary

[0105]

[0106] Example 2 Determination of C5a-blocking cyclic peptide (Cp) binding specificity

[0107] This study investigated whether the specific blockade of Cp against C5a-C5aR1 would affect the interaction between C5a and C5aR2. Human neutrophils (Ausnutria Biotech, China) were sorted by flow cytometry and incubated at room temperature for 0.5 h with 5 μg C5aR1 antibody (BioLegend, USA), 5 μg C5aR2 antibody (BioLegend, USA), 10 μg Cp, Cp (10 μg) + C5aR1 antibody (5 μg), or Cp (10 μg) + C5aR2 antibody (5 μg), respectively. Then, they were incubated with 10 ng of Cy5 (10 ng / 100 μL) labeled human C5a (MCE, China) at 4 °C in the dark for 0.5 h. The samples were then centrifuged at 300 g for 5 min, washed twice with PBS, and the fluorescence intensity of Cy5 in each group was detected by flow cytometry.

[0108] Figure 3 The results show the flow cytometry semi-quantitative results of C5a blocking cyclic peptide Cp specifically blocking the C5a-C5aR1 interaction. The blank group (Blank group) consisted of cells supplemented with only 1640 complete medium (Gibco); the control group (Control group) consisted of cells supplemented with 1640 complete medium containing Cy5-C5a. Figure 3 The results showed that the fluorescence signal in the Cp group was significantly lower than that in the Control group, and the fluorescence signal in the Cp+C5aR1 antibody group was further downregulated compared to the Cp group. However, the fluorescence signal in the Cp+C5aR2 antibody group was essentially the same as that in the Cp group, demonstrating that Cp specifically blocked the C5a-C5aR1 interaction without affecting the C5a-C5aR2 interaction. Data are expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001. ns indicates no significant difference.

[0109] Example 3 Determination of C5a-blocking cyclic peptide (Cp) binding specificity

[0110] This study investigated whether Cp binding to C5a affected C5 lysis and MAC formation. Hemolysin (Borsi, China) was mixed with 2% sheep erythrocytes (Borsi, China) at a 1:1 volume ratio and incubated at 37°C for 30 minutes. After washing with 5 volumes of PBS, the mixture was centrifuged at 1000 rpm for 10 minutes and resuspended in PBS to obtain sheep erythrocytes conditioned with 2% hemolysin. The sheep erythrocytes completely lysed in distilled water served as a positive control (100% hemolysis rate), and the PBS group served as a negative control (0% hemolysis rate). Serum from 8-10 week old male C57BL / 6 normal mice (Jicui Yaokang) was serially diluted at 0, 1, 2, 4, 8, 16, 32, 64, and 128-fold. 50 μL of sheep erythrocytes conditioned with 2% hemolysin were mixed with distilled water, PBS, and serially diluted mouse serum at a volume ratio of 1:1 and incubated at 37°C for 30 min. After centrifugation at 1000 rpm for 10 min, the absorbance of the supernatant was measured at 540 nm using a multimode microplate reader (BioTek, USA). The amount of mouse plasma required for 50% lysis of erythrocytes was determined.

[0111] Mouse plasma (the volume required for 50% lysis of red blood cells) was incubated with serially diluted (10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL) Cp or C5 antibody (eculizumab, MCE, China) at 37°C for 30 minutes, with C5 antibody serving as a positive control. Subsequently, sheep red blood cells conditioned with 2% hemolysin were added to the reaction system, incubated at 37°C for 30 minutes, centrifuged at 1000 rpm for 10 minutes, and the absorbance of the supernatant at 540 nm was measured using a microplate reader. The corresponding hemolysis percentage was calculated as follows: Hemolysis percentage = (OD of the sample to be tested) / (OD of the sample to be tested). 540 nm Value / OD of distilled water group 540nm (Value) × 100%.

[0112] Figure 4 The results show the hemolysis assay results of C5a blocking the specific binding of the cyclic peptide Cp to C5a. The results show that almost no hemolysis occurred in the C5 antibody group, while the hemolysis rate of the Cp group at different concentrations was close to 100%, similar to the distilled water group. This proves that Cp specifically binds to C5a and does not hinder C5 cleavage or the formation of the MAC membrane attack complex by C5b-9.

[0113] Example 4: Determination of plasma stability of C5a-blocking cyclic peptide (Cp)

[0114] A cecal ligation and puncture (CLP) model was established using 8-10 week old male C57BL / 6 mice. In short, under general anesthesia, mice underwent a midline laparotomy, and the distal three-quarters of the cecum was ligated with sutures. Subsequently, a 22 μg needle was used to puncture the ligated segment, expelling a small amount of fecal contents. The cecum was then repositioned into the abdominal cavity, and the laparotomy incision was closed in layers. Mice were subcutaneously given 1 mL of sterile saline for postoperative fluid resuscitation. A sham-operated control group underwent the same surgical procedure but without cecal ligation or puncture. Twenty-four hours after CLP, whole blood was collected from the CLP model mice using an anticoagulated capillary orbital sampling method. The blood was centrifuged at 1000 g for 10 minutes, and the plasma was used for cyclic peptide Cp plasma stability testing.

[0115] 100 μg of Cp or Lp (the linear form of Cp) was incubated with 50% (v / v) plasma from CLP model mice at 37°C for 24 hours to assess the plasma stability of the cyclic peptide Cp, with Lp serving as a control. Cyclic peptide Cp was extracted from plasma using 15% TCA (Sinopharm Reagent, China) at a plasma-to-TCA ratio of 5:1. After vortexing for 1 minute, precipitation was performed at 4°C for 20 minutes, followed by centrifugation at 12000 rpm for 10 minutes. 5 μL of the supernatant was collected for quantification using high-performance liquid chromatography (Agilent 1260, USA). Mobile phase A was 0.05% (w / w) trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile solution.

[0116] Figure 5 The figure shows the HPLC quantitative results of plasma stability of the C5a-blocked cyclic peptide Cp in CLP model mice. The results show that in the plasma of CLP model mice with high protease expression, the residual amount of the cyclic peptide Cp of this invention was still 90% of the initial amount after 24 hours, demonstrating that Cp has good plasma stability and is suitable for intravenous administration for the prevention and treatment of sepsis.

[0117] Example 5 Evaluation of the inhibitory effect of C5a blocking cyclic peptide (Cp) on plasma inflammatory factors and chemokines in CLP-induced sepsis model mice.

[0118] Eight to ten-week-old C57BL / 6 mice were randomly divided into three groups: a sham-operated group (Sham group), a CLP-induced sepsis mouse model group (CLP group), and a C5a-blocking cyclic peptide Cp treatment group (CLP+Cp group), with four mice in each group. The specific method for establishing the mouse CLP model is described in Example 4. One hour after surgery, each group of mice was intravenously injected with 100 μL of physiological saline, physiological saline, or Cp (5 mg / kg mouse body weight, dissolved in physiological saline), respectively. Twenty-four hours after CLP modeling, 500 μL of whole blood was collected using an anticoagulant capillary orbital blood sampling method. Plasma was separated by centrifugation at 1000 g for 10 minutes, and plasma inflammatory factors and chemokines were quantified using an ELISA kit (Huamei Biotechnology, China). The inhibition rate of inflammatory factors or chemokines was calculated with a recovery to the sham-operated group level as 100%.

[0119] Figure 6 , Figure 7 The image shows the ELISA quantitative results of the inhibitory effect of C5a-blocking cyclic peptide Cp on plasma inflammatory factors in a CLP-induced sepsis model mouse. IL-6 and TNF-α are core cytokines driving the cytokine storm in sepsis. Figure 6 , Figure 7 As can be seen, 24 hours after CLP surgery, compared with the untreated CLP group, the levels of inflammatory factors IL-6 and TNF-α in the plasma of CLP model mice in the Cp treatment group (CLP+Cp group) were significantly downregulated, demonstrating that Cp can effectively inhibit inflammatory factors in the plasma of sepsis model mice. Data are expressed as mean ± standard deviation. An asterisk indicates a statistically significant difference between groups: *p<0.05, **p<0.01, ***p<0.001.

[0120] Figure 8 , Figure 9 The image shows the ELISA quantitative results of the inhibitory effect of C5a-blocking cyclic peptide Cp on plasma chemokines in a CLP-induced sepsis model mouse. CXCL1 and CCL2 are important chemokines in sepsis, with CXCL1 being a key chemokine for neutrophils and CCL2 a key chemokine for monocytes / macrophages. CXCL1 and CCL2 exacerbate the inflammatory response by recruiting and activating immune cells (such as neutrophils, monocytes / macrophages). Figure 8 , Figure 9 It can be seen that 24 hours after CLP surgery, compared with the untreated CLP group, the levels of chemokines CXCL1 and CCL2 in the plasma of CLP-treated mice (CLP+Cp group) were significantly downregulated, demonstrating that Cp can effectively inhibit chemokines in the plasma of sepsis-induced mice. Data are expressed as mean ± standard deviation. An asterisk indicates a statistically significant difference between groups: *p<0.05, **p<0.01, ***p<0.001.

[0121] Example 6 Evaluation of the inhibitory effect of C5a blocking cyclic peptide (Cp) on C5a-induced chemotaxis in mouse and human neutrophils.

[0122] 100 ng of mouse C5a protein (ACROBiosystems, China) or 100 ng of human C5a protein (ACROBiosystems, China) were pre-incubated with specified concentrations (10 ng, 100 ng, or 1 μg) of cyclic peptide Cp in 1640 complete medium for 30 minutes. Human neutrophils (Ausnutria Biotech, China) were sorted by flow cytometry for chemotaxis experiments. 1×10 4 Cells at a specific density were added to the upper chamber of a 96-well cell chemotaxis plate (Sartorius, Germany) at a volume of 60 μL. The lower chamber contained 200 μL of 1640 complete culture medium containing C5a and different concentrations of Cp. The experiment was divided into four groups: Blank (blank control), C5a, C5a + Cp (1 μg), C5a + Cp (100 ng), and C5a + Cp (10 ng). The Blank group had a lower chamber containing 1640 complete culture medium, while the C5a group had a lower chamber containing 100 ng of C5a added to 1640 complete culture medium. The lower chambers of the C5a+Cp (1 μg), C5a+Cp (100 ng), and C5a+Cp (10 ng) groups contained 100 ng of C5a and different concentrations of Cp (1 μg / 200 μL, 100 ng / 200 μL, and 10 ng / 200 μL), respectively, in 1640 complete culture medium. Each group was incubated at 37°C for 24 hours. The results were obtained using IncuCyteZOOM. ® A live-cell imaging system (Sartorius, Germany) was used to quantitatively analyze the chemotactic response of neutrophils to C5a with and without the presence of C5a-blocking peptides. The vertical axis, "Ratio of real-time total cell area to initial area," represents the ratio of the real-time total cell area in a specified field of view to the initial area at 0 hours, reflecting cell migration. A larger ratio indicates a smaller proportion of migrating cells.

[0123] Figure 10 , Figure 11 The figures show the quantitative results of the inhibitory effect of the C5a-blocking cyclic peptide Cp on C5a-induced chemotaxis in mouse and human neutrophils. Figure 10 It can be seen that, 24 hours after chemotaxis, the ratio of "real-time total cell area / initial area" in the groups co-incubated with C5a at different concentrations of Cp (1 μg, 100 ng, and 10 ng) was significantly higher than that in the C5a group, demonstrating that the cyclic peptide Cp can effectively inhibit C5a-induced chemotaxis in mouse neutrophils. Figure 11As can be seen, 24 hours after chemotaxis, the ratio of "real-time total cell area / initial area" in the groups co-incubated with C5a at different concentrations of Cp (1 μg, 100 ng, and 10 ng) was significantly higher than that in the C5a group, demonstrating that the cyclic peptide Cp can effectively inhibit C5a-induced chemotaxis in human neutrophils. Data are expressed as mean ± standard deviation. An asterisk indicates a statistically significant difference between groups: *p<0.05, **p<0.01, ***p<0.001.

[0124] Example 7: Effect of C5a blocking cyclic peptide (Cp) treatment on the survival of a sepsis model mouse.

[0125] Eight to ten-week-old C57BL / 6 mice were randomly divided into three groups: a sham-operated group (Sham group), a CLP-induced sepsis mouse model group (CLP group), and a C5a-blocking cyclic peptide Cp treatment group (CLP+Cp group), with 10 mice in each group. The method for establishing the mouse CLP model is described in Example 4. One hour after CLP modeling, the mice in each group were injected intravenously with 100 μL of physiological saline, physiological saline, or Cp (5 mg / kg mouse body weight, dissolved in physiological saline), respectively. Animal mortality was determined by confirming animal death. The survival of mice in each group was observed for one week, and Kaplan-Meier survival curves were plotted.

[0126] Figure 12 The image shows the Kaplan-Meier survival curves of mice treated with C5a-blocking cyclic peptide Cp. The results showed that the median survival (LC50) of untreated CLP-treated mice was 36 hours, while that of mice treated with Cp was 120 hours. This indicates that C5a-blocking cyclic peptide Cp significantly prolonged the survival of CLP-induced sepsis model mice. An asterisk (*p<0.05, **p<0.01, ***p<0.001) indicates statistically significant differences between groups.

Claims

1. A cyclic peptide that specifically recognizes complement C5a, characterized in that, The amino acid sequence of the cyclic peptide is shown in SEQ ID NO:1, with a lactam bridge introduced between serine at position 1 and phenylalanine at position 12.

2. A modified polypeptide fragment, characterized in that, The amino acid sequence of the modified polypeptide fragment is shown in SEQ ID NO: 1, and the side chain protecting groups of Ser, His, Glu and Arg are tBu, Trt, OtBu and Pbf, respectively.

3. The use of the modified polypeptide fragment as described in claim 2 in the preparation of a cyclic peptide that specifically recognizes complement C5a.

4. A method for synthesizing a cyclic peptide that specifically recognizes complement C5a, characterized in that, The synthesis method includes the following steps: (a) Using resin as a solid support, amino acid residues are coupled sequentially from the C-terminus to the N-terminus according to the sequence of SEQ ID NO: 1, wherein the side chain protecting groups of Ser, His, Glu and Arg are tBu, Trt, OtBu and Pbf, respectively; (b) After the linear peptide synthesis is completed, the straight-chain peptide is cleaved from the resin by lysis buffer A to obtain a straight-chain peptide with side chain protecting groups; (c) Intramolecular cyclization of the product of step (b) in the presence of a condensation system; and, (d) After cyclization, the side chain protecting groups were removed using lysis buffer B to obtain the cyclic peptide shown in SEQ ID NO:

1.

5. The synthesis method as described in claim 4, characterized in that, The synthesis method satisfies one or more of the following: 1) In step (b), the lysis buffer A comprises TFA and DCM; 2) Step (c), the condensation system contains PyBop and DIEA; and, 3) Step (d): The lysis solution B contains TFA, TIS and H2O.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the cyclic peptide as described in claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

8. A reagent or kit, characterized in that, The reagent or kit contains the cyclic peptide as described in claim 1 or the pharmaceutical composition as described in claim 6.

9. The use of the cyclic peptide as described in claim 1 in the preparation of a medicament for treating sepsis.

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