Novel complement inhibitor as well as preparation, application and product thereof

Abstract

The invention provides a novel complement inhibitor as well as preparation, application and a product thereof, and belongs to the field of molecular drugs, a complement inhibition active part comprises a cyclic peptide and a pharmaceutically acceptable salt thereof, and the cyclic peptide is shown as a general formula I: R1-ICXaa3-Xaa4-QD-Xaa7-G-Xaa9-HRCT-R2, wherein Xaa7 and Xaa9 are selected from extraordinary amino acids, and Xaa3 and Xaa4 comprise at least one extraordinary amino acid. When the cyclopeptide provided by the invention is used as an active component of the complement inhibitor, the cyclopeptide has relatively good affinity to C3 protein, has relatively good pharmacokinetic properties, and has a very good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of molecular medicine, and in particular relates to a novel complement inhibitor and its preparation, application and product. Background Art

[0002] Complement is a group of globulins that exist in the blood, tissue fluid and cell membrane surface of humans and animals and have enzyme-like activity after activation. The complement system is an important component of innate immunity and participates in human immune regulation and immune injury response. The complement system is composed of more than 30 soluble proteins and membrane-bound proteins. Its activation pathways mainly include the classical pathway, the alternative pathway and the lectinactivation pathway. Complement exists in plasma in an inactive state and has biological activity after activation. It can clear immune complexes by lysis, conditioning, phagocytosis and mediating inflammatory responses, showing corresponding biological functions.

[0003] The three activation pathways of the complement system are all centered on the formation of C3 convertase and C5 convertase. The corresponding biologically active fragments are generated by cleaving C3 and C5, further amplifying the complement reaction. These fragments modify the target surface and promote processes such as phagocytosis, inflammation, and immune regulation. Among them, the front-end reaction of these activation pathways can be regarded as the front-end reaction from contact with the activation initiator to the generation of C3 convertase (and cleavage of C3). Although the front-end reactions of these activation pathways are different, they have a common terminal pathway, that is, the generation of C5 convertase, which forms C5b fragments by cleaving C5, and reacts with C6, C7, C8, and C9 in sequence, and finally forms a membrane attack complex (MAC) to exert a cytolytic effect.

[0004] Although the complement system plays an important role in human immunity, inappropriate or excessive complement activation is the root cause or contributing factor of many serious diseases. The discovery of complement inhibitors provides a good research direction for the treatment of related diseases. There are complement drugs in the fields of diseases such as paroxysmal nocturnal hemoglobinuria and geographic atrophy caused by age-related macular degeneration. According to the three activation pathways of the complement system, it can be known that inhibiting C3 can control the front-end reaction of the activation pathway, so the development of C3 complement inhibitors has a good application prospect. C3, as the complement protein with the highest content in human serum, is composed of two chains, α and β. The activated C3 is cleaved into fragments with equally important biological activities: C3a, C3b, iC3a, C3d, etc. C3 contains 41 exons and has a rich structure that provides multiple different ligand binding sites, such as the conserved domains disclosed in the prior art: α2 macroglobulin domain (A2M), C3a anaphylatoxin domain, etc. (Zan Qi, Liu Xin, Pang Yue, et al. Progress in research on the structure and function of complement C3 [J]. Chinese Journal of Immunology, 2014(04):549-553.DOI:10.3969 / j.issn.1000-484X.2014.04.031.), which is very important in immune surveillance and immune response pathways.

[0005] Compstatin is the first discovered C3 complement inhibitor. It is a 13-cyclic peptide that can bind to both C3a and C3b and prevent C3 convertase from cleaving native C3. The cyclic peptide is a molecule composed of a polar part and a non-polar part. The polar part includes a type I β turn and the non-polar part includes a disulfide bond. The prior art has verified that the four residues of the β turn and the disulfide bond with a surrounding hydrophobic cluster play an important role in the inhibitory activity of C3 compstatin. The prior art has continuously optimized the earliest disclosed compstatin structure to improve its pharmaceutical properties, such as the optimized compstatin analog Ac-ICVW(CH3)QDWGAHRCT-NH2(CC), which is 16 times more active than the parent, as described in "Morikis D, Soulika AM, Mallik B, et al. Improvement of the anti-C3 activity of compstatin using rational and combinatorial approaches".

[0006] Empaveli (pegcetacoplan) is the first C3 complement inhibitor drug to be marketed. It is a synthetic cyclic peptide coupled to a polyethylene glycol polymer that can specifically bind to C3a and C3b. Empaveli's currently approved indications include paroxysmal nocturnal hemoglobinuria (PNH) and geographic atrophy caused by dry maculopathy.

[0007] Other optimization methods disclosed in the prior art of compstatin include methylation on the peptide backbone and substitutions on the flanking positions, and a series of compstatin analogs with improved potency have been developed (Qu et al., 2011, Molec Immunol 48:481-489, WO 2010 / 127336). These modifications have produced compstatin analogs with binding affinities superior to most active analogs reported to date.

[0008] A Chinese patent with application number CN201980030725.6 discloses a compstatin analog with a C-terminal and / or N-terminal modification, which improves the solubility of the peptide without significantly reducing its pharmacokinetic properties and, in some cases, confers enhanced plasma and vitreous stability and / or binding affinity to C3 and its fragments, but does not actually improve the properties of the cyclic peptide itself.

[0009] Some optimization methods also use the optimization of the types of amino acids on the peptide, but there are various ways to optimize amino acids, and the results of any optimization method are not certain. Given the therapeutic potential of compstatin analogs in various diseases, it is very important to further optimize compstatin to obtain greater efficacy. Summary of the invention

[0010] In order to solve the above problems, the present invention provides a novel complement inhibitor and its preparation, application and product.

[0011] the term:

[0012] In the present invention, "complement inhibitors" are sometimes also referred to as compstatins, complement inhibition analogs, and compstatins are cyclic peptides that bind to the complement component C3 and inhibit complement activation. Compstatins inhibit the cleavage of C3 into C3a and C3b by convertase. Since C3 is a central component of all three complement activation pathways, compstatins and their analogs can inhibit the activation of proteins that converge all three pathways. The ability of compstatins and their analogs to inhibit the alternative pathway of complement activation can significantly contribute to their efficacy in certain ocular conditions described herein.

[0013] In the present invention, "cyclic peptide" generally refers to a cyclic polypeptide, which refers to a polypeptide chain formed by chemical bonds between protein or non-protein amino acids in a cyclic sequence, which can generally be formed by disulfide bonds, amide bonds, or other cyclic forms disclosed in the prior art. There may be multiple cyclic forms in the same cyclic peptide.

[0014] As used herein, "polypeptide" refers to an amino acid polymer, optionally including one or more amino acid analogs. Proteins are molecules composed of one or more polypeptides. Peptides are relatively short polypeptides, typically about 2 to 60 amino acids in length. The terms "protein", "polypeptide" and "peptide" are used interchangeably. The polypeptides used herein may contain amino acids, such as amino acids naturally occurring in proteins, amino acids that are not naturally occurring in proteins, and / or amino acid analogs of non-amino acids. Amino acid "analogs" used herein may be different amino acids that are structurally similar to amino acids, or compounds other than amino acids that are structurally similar to amino acids. A large number of industry-recognized analogs of the 20 amino acids commonly found in proteins are known. One or more amino acids in a polypeptide may be modified, for example, by adding chemical entities (e.g., carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, conjugated linkers), functionalization or other modifications, etc.

[0015] In the present invention, some abbreviations are explained as follows:

[0016]

[0017]

[0018] In the present invention, abbreviations not specifically noted refer to the current common abbreviations, specifically, the abbreviations of common amino acids.

[0019] In the present invention, the scope of "uncommon amino acids" includes all amino acid types except the 20 common amino acids known in the industry. These include natural amino acids and unnatural amino acids. Natural amino acids can be amino acids that are naturally present in nature and have been discovered or not yet discovered. Unnatural amino acids can be artificially modified amino acids.

[0020] In the present invention, artificial modification methods of "non-natural amino acids" include, but are not limited to, phosphorylation, methylation, acetylation, ubiquitination, glycosylation, etc. Artificially modified amino acids prepared by conventional amino acid modification methods in the art all fall within the scope of non-natural amino acids covered by the present invention.

[0021] The invention first provides a novel complement inhibitor.

[0022] The active portion of the complement inhibitor comprises a cyclic peptide or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide is represented by the general formula I:

[0023] General formula I: R1-IC-Xaa3-Xaa4-QD-Xaa7-G-Xaa9-HRCT-R2(CC);

[0024] Among them, Xaa7 and Xaa9 are selected from uncommon amino acids and derivatives, Xaa3 and Xaa4 include at least one uncommon amino acid and derivative, and R1 and R2 are modifying groups.

[0025] Specifically, Xaa3 and Xaa4 may each independently include one uncommon amino acid, or Xaa3 may be selected from uncommon amino acids and Xaa4 may be selected from common amino acids; or Xaa4 may be selected from uncommon amino acids and Xaa3 may be selected from common amino acids.

[0026] The uncommon amino acids in the general formula may be: tryptophan (W) analogs, alanine (A) analogs, aminobutyric acid analogs, aminoisobutyric acid analogs, 4-aminopiperidine-4-carboxylic acid analogs, benzoylphenylalanine analogs.

[0027] The modification of the analogs can be methylation, acetylation, hydroxylation, carboxylation, phosphorylation, naphthylation, nitrogen atom hybridization, fluorination, acylation, alkoxylation, or halogenation.

[0028] Preferably, the tryptophan analog is selected from: 5-fluoro-DL-tryptophan, azatryptophan or N-methyltryptophan.

[0029] Preferably, when Xaa3 is selected from uncommon amino acids, it may be Abu;

[0030] Preferably, when Xaa3 is selected from common amino acids, it can be V or Y.

[0031] Preferably, when Xaa4 is selected from uncommon amino acids, it can be any one of 2Nal, W(CH3), and Bpa.

[0032] In some preferred conditions, the Xaa4 must be selected from uncommon amino acids.

[0033] Preferably, the Xaa7 is selected from 2Nal or 5F-Trp.

[0034] Preferably, the Xaa9 is selected from Aib or Pip.

[0035] In some specific embodiments, Xaa3, Xaa4, Xaa7, and Xaa9 are each independently selected from the above preferred conditions, including but not limited to:

[0036] (1) In the cyclic peptide, Xaa3 is selected from V or Y, and Xaa4 is selected from any one of 2Nal, W(CH3), and Bpa;

[0037] Or in the cyclic peptide described in (2), Xaa4 is selected from any one of 2Nal, W(CH3), and Bpa, and Xaa7 is selected from any one of 2Nal and 5F-Trp;

[0038] Or in the cyclic peptide described in (3), Xaa4 is selected from any one of 2Nal, W(CH3), and Bpa, and Xaa9 is selected from any one of Aib and Pip;

[0039] or (4) wherein Xaa3 is Abu, and Xaa7 is selected from 2Nal or 5F-Trp;

[0040] or (5) wherein Xaa3 is Abu, and Xaa9 is selected from either Aib or Pip;

[0041] Or in the cyclic peptide described in (6), Xaa3 is Abu, Xaa4 is selected from any one of 2Nal, W(CH3), and Bpa, Xaa7 is selected from any one of 2Nal and 5F-Trp, and Xaa9 is selected from any one of Aib and Pip;

[0042] Or in the cyclic peptide described in (7), Xaa3 is selected from V or Y, Xaa4 is selected from any one of 2Nal, W(CH3), Bpa, Xaa7 is selected from any one of 2Nal or 5F-Trp, and Xaa9 is selected from any one of Aib or Pip.

[0043] The R1 or R2 can be selected from reactive functional groups, including but not limited to the following groups: olefins, acetylene, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, diazos, nitro, nitrile, thiols, sulfides, disulfides, sulfur oxides, sulfones, sulfonic acids, sulfuric acids, acetals, ketals, anhydrides, sulfates, sulfenic acids, isonitriles, amidines, imides, imide esters, nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamic acid, allene, orthoesters, sulfites, enamines, alkynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imides, azides, azo compounds, azoxy compounds and nitroso compounds. Reactive functional groups also include groups frequently used in the preparation of bioconjugates, such as N-hydroxysuccinimide esters, maleimide, sulfhydryls, and the like.

[0044] Preferably, the R1 may include one or more of hydrogen, acetyl, methoxy, PEG, and carboxyl.

[0045] Preferably, the R2 may include an amino group.

[0046] Preferably, the R1 or R2 comprises an amino acid or a variant thereof.

[0047] In some embodiments, said R1 or R2 independently comprises tyrosine or isoleucine.

[0048] In some embodiments, the R1 comprises isoleucine and the R2 comprises tyrosine.

[0049] Preferably, R1 and R2 form an amide bond to form a ring from head to tail of the polypeptide.

[0050] In some embodiments, the complement inhibitor activity portion is selected from any one or more of SEQ ID NO.1-6:

[0051] SEQ ID NO.1: Ac-ICV-W(CH3)-QD-2Nal-G-Aib-HRCT-NH2(CC);

[0052] SEQ ID NO.2: Ac-ICV-AzaTrp-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0053] SEQ ID NO.3: Ac-ICV-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0054] SEQ ID NO.4: Ac-IC-Abu-W(CH3)-QD-5F-Trp-G-Pip-HRCT-NH2(CC);

[0055] SEQ ID NO.5: Ac-IC-Abu-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0056] SEQ ID NO. 6: Ac-IC-Y-Bpa-QD-5F-Trp-G-Aib-HRCT-NH2(CC).

[0057] Under some conditions, the general formula I has a structure similar to compstatin:

[0058] Cyclization is carried out through a disulfide bridge formed by Cys-2 and Cys-12. Cys-2 represents cysteine ​​(C) at position 2 in the general formula, and Cys-12 represents cysteine ​​(C) at position 12 in the general formula.

[0059] The complement inhibitor may directly include any one or more of the aforementioned cyclic peptides;

[0060] The complement inhibitor may include the product of the above-mentioned cyclic peptide after side chain modification, and the side chain modification may be a small molecule protein modification or a C-terminal modification.

[0061] In the present invention, the "side chain modification" may be referred to as "side chain modification" or "branch chain modification" in some cases. As known to those skilled in the art, the termini of the peptide are by default the free amino group at the N-terminus and the free carboxyl group at the C-terminus. The peptide termini often need to be blocked, i.e., acetylation at the N-terminus and amidation at the C-terminus. This modification avoids the introduction of excess charge and also makes it more resistant to exonuclease action, making the peptide more stable.

[0062] In the present invention, the small molecule proteins used for modification can be achieved by covalent modification, and can be inorganic small molecules or organic small molecules, such as phosphorylation, glycosylation, acylation, etc.

[0063] The complement inhibitor may also be an optimized product obtained by conventional technical means of the general formula I, without changing the activity of the cyclic peptide itself. The optimization may occur on R1 or R2, or on the amino acids of the main chain. Such as PEG optimization, conjugation, homopolymers, heteropolymers, amino acid chirality optimization, hydrophilic / charged residue incorporation, increased transmembrane peptides, N-methylation in the chain, etc. As a preferred embodiment, Xaa9 may include an N-methyl derivative. The general formula I or its optimized form may be regarded as a compstatin analog.

[0064] In the present invention, the "methylation" refers to the process of catalytically transferring methyl groups from active methyl compounds to other compounds, which can form various methyl compounds, or chemically modify certain proteins to form methylated products. Specifically, the cyclopeptide provided by the present invention is chemically modified to form a methylated product.

[0065] In the present invention, the "N-methylation in the chain" refers to the methylation occurring on any one or more amino acids in the chain, specifically, a methyl group is connected to -NH2 of any one or more amino acids in the chain to form an -NH-CH3 structure. Preferably, the N-methylation in the chain occurs on any one amino acid in the chain. In some embodiments, the N-methylation occurs on the 9th amino acid of the cyclic peptide described in the present application.

[0066] In the present invention, the "amino acid chirality" refers to a molecule in an amino acid that is different from its mirror image and cannot overlap with each other and has a certain configuration or conformation. Chirality is used to express the relationship between an optically active molecule and a three-dimensional image whose mirror image cannot overlap. All chiral molecules are optically active, and all molecules of optically active compounds are chiral molecules. Chiral molecules include asymmetric molecules without any symmetry factors and asymmetric molecules with a simple symmetry axis but no other symmetry factors.

[0067] The complement inhibitor can be a cell-penetrating peptide added to the general formula I. The cell-penetrating peptide can be a cell-penetrating peptide disclosed in the prior art, or an amino acid sequence that has not been disclosed in the prior art but still has the same function as the cell-penetrating peptide in the prior art after disclosure.

[0068] As known to those skilled in the art, cell-penetrating peptides (CPPs), also known as transduction peptides, are a class of short peptide molecules with the ability to penetrate the cell membrane, which can enter the interior of the cell and interact with the target molecule through mechanisms such as membrane perturbations and membrane flipping. Cell-penetrating peptides can play a role in drug transport. Cell-penetrating peptides are usually composed of 3-30 amino acids and have characteristics such as positive charge or hydrophobicity. These properties enable them to interact with negatively charged components (such as phosphatidylinositol, etc.) on the cell membrane and enter the interior of the cell. Cell-penetrating peptides also have good biological stability, repeatable synthesis and change. In the present invention, as a cell-penetrating peptide for optimizing general formula I to obtain a complement inhibitor, its specific type is not limited, and it can be composed of 3-30 amino acids or a composition of an amino acid number outside the range. Generally, the cell-penetrating peptide can be 2-10, 2-12, 2-18, 2-20, 2-25, 5-10, 5-20, 5-25, 10-15, 10-20, 10-25, 15-20, 15-25, 15-30, 20-30, 25-30, 13-19 or 18-24 amino acids in length. In other embodiments, the cell-penetrating peptide can be modified, such as cell-penetrating peptide TAT: H2N-YGRKKRRQRRR-OH.

[0069] The incorporation of hydrophilic / charged residues can be used as a method to increase the solubility of cyclic peptides. Therefore, the complement inhibitor can be a hydrophilic / charged residue incorporated into the general formula I, and the number can be two or three. The incorporation of the hydrophilic / charged residue can occur at the C-terminus or the N-terminus or simultaneously. Specifically, in the general formula I, it can occur on R1 or R2 of the general formula I or simultaneously. The simultaneous occurrence refers to the incorporation of hydrophilic / charged residues on both R1 and R2. In some cases, it can be determined that the solubility of the peptide increases with the increase in the number of lysine residues, but it does not mean that this modification has the same effect in all cases. Residues that can be incorporated into the general formula I include KK, KKK, KRK, and RRR. Adding two or three lysine residues can improve the solubility of the cyclic peptide in some cases, and while maintaining the inhibitory ability, it also improves the binding affinity, exhibits improved pharmacokinetic characteristics, expands the range of administration routes, reduces the frequency of administration during chronic treatment, and improves drug compliance. This has been disclosed in some prior arts, such as “Nadja B, Tchilabalo A, Resuello R, et al. New analogs of the complement C3 inhibitor compstatin withincreased solubility and improved pharmacokinetic profile[J]. Journal of Medicinal Chemistry, 2018: acs.jmedchem.8b00560.”

[0070] Preferably, the incorporation of the hydrophilic / charged residue occurs on R2, specifically, before the amino group. More specifically, any one or more K or R are connected before the amino group. Optionally, one K or R can be connected; KK, RR, KR or RK can be connected; three amino acid residues can also be connected, wherein each amino acid residue is independently K or R, such as any one of KKK, KKR, KRK, RKK, KRR, RKR, RRK, and RRR.

[0071] The complement inhibitor may be a cyclic peptide of the general formula I, comprising a clearance reducing portion having a molecular weight of 1 kD to 100 kD, specifically, the clearance reducing portion comprises polyethylene glycol (PEG). This optimization does not affect the inhibitory activity of the cyclic peptide itself, and is a conventional optimization method in the art.

[0072] Preferably, the molecular weight of the clearance reducing portion can be 20-80kD, 20-90kD, 30-80kD, 30-90kD, 40-80kD, 40-50kD, 50-60kD, 70-80kD, 70-90kD, 80-90kD.

[0073] Preferably, the PEG can be a branched PEG or a linear PEG; further preferably, the clearance reducing moiety comprises a branched PEG having 3-10 branches, and at least about 50% of the branches have a compstatin analog moiety attached thereto.

[0074] Still further, the clearance reducing moiety comprises a branched PEG having 3-4, 3-6, 3-9, 4-5, 4-7, 4-9, 5-6, 5-8, 6-7, 6-9, 7-8, 7-9, or 8-9 branches; and at least about 55%, 58%, 60%, 64%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 98%, or 95% of the branches have a compstatin analog moiety attached thereto.

[0075] By adding molecules (such as polyethylene glycol or similar molecules), it is possible to modify, optionally linked to a cell-reactive portion or a targeting portion, to stabilize the compound, reduce its immunogenicity, prolong its life in vivo, enhance or reduce its solubility and / or enhance its resistance to degradation. Pegylation methods are well known in the art. Pegylation is the covalent coupling of activated PEG to a protein or polypeptide molecule by chemical methods. Complement inhibitors can be pegylated at the C-terminus or N-terminus or both, specifically, polyethylene glycol molecules are added to R1 or R2 of Formula I, or polyethylene glycol molecules can be added to both R1 and R2. Low molecular weight PEG chains can also be attached to the C-terminus or Lys residues of compstatin as disclosed in the prior art to develop new analogs of the C3 inhibitor compstatin. Compared with the parent peptide, the 3kD PEG attached can significantly improve the solubility at physiological pH, while maintaining the inhibitory activity and showing a better pharmacokinetic profile. PEGylation is a relatively mature half-life extension technology in clinical practice. Its safety has been fully verified in many years of clinical practice and has been approved by drug regulatory authorities in most countries / regions for intravenous, oral and cutaneous use in humans.

[0076] In some embodiments, it is known to those skilled in the art that the solubility of the peptide can be enhanced by modifying the terminal region of the cyclic peptide, such as PEG modification, but not limited to PEG modification. Under some conditions, those skilled in the art can know that in some cases the size of the PEG chain is directly related to the solubility of the compound, but this does not mean that this is the case in all cases, and there may be other modifications that affect the solubility of the compound.

[0077] Preferably, the PEG modification occurs on R1 and / or R2, and may also include any one or both of a carboxyl group and a methoxy group, and the PEG modification is freely combined with the carboxyl group or the methoxy group.

[0078] Preferably, the R2 of the general formula I after PEG modification may have a structure similar to the following: -COCH2-PEG-OMe; the molecular weight of PEG may be 1 kD to 100 kD, referring to the part on reduction of clearance rate.

[0079] In another embodiment, the cyclic peptide is fused to the Fc domain of an immunoglobulin or a portion thereof. In some other embodiments, the cyclic peptide is conjugated to an albumin portion or an albumin binding peptide. Thus, in certain embodiments, the cyclic peptide is modified with one or more polypeptide or non-polypeptide components, for example, the cyclic peptide is pegylated or conjugated to another portion. In certain embodiments, the component is not an Fc domain of an immunoglobulin or a portion thereof. The cyclic peptide can be provided as part of a multimer or supramolecular complex, which can include a single molecule substance or a plurality of different substances.

[0080] In certain embodiments, the complement inhibitor is a multivalent compound comprising a plurality of cyclic peptides covalently or non-covalently linked to a polymeric backbone or scaffold. The linked individual cyclic peptides may be the same or different. In certain embodiments of the invention, the cyclic peptide comprises multiple instances or copies of a single compstatin analog portion. In other embodiments of the invention, the multivalent compound comprises one or more instances of each of two or more different cyclic peptides (e.g., 3, 4, 5 or more different cyclic peptides). In certain embodiments of the invention, the number of cyclic peptides may be 1-10. In other embodiments of the invention, the number of cyclic peptides may be 10-20, 20-100, 100-1000, 1000-10000, 10000 to 50000, 50000-100000, 100000 to 1000000.

[0081] The cyclic peptide can be directly connected to the polymeric scaffold, or can be connected through a linking moiety that connects the cyclic peptide to the polymeric scaffold. The linking moiety can be connected to a single cyclic peptide and to the polymeric scaffold. Alternatively, the linking moiety can have multiple cyclic peptides connected to it, so that the linking moiety connects multiple cyclic peptides to the polymeric scaffold.

[0082] In certain embodiments, the cyclic peptide comprises an amino acid, such as a Lys residue, having a side chain containing a primary or secondary amine. For example, a Lys residue or a sequence comprising a Lys residue is attached to the N-terminus and / or C-terminus of the cyclic peptide. In certain embodiments, the Lys cyclic peptide cyclic portion is separated by a rigid or flexible spacer. The spacer may, for example, comprise a substituted or unsubstituted, saturated or unsaturated alkyl chain, a oligo(ethylene glycol) chain and / or other moiety. The length of the chain may be, for example, 2-20 carbon atoms. In other embodiments, the spacer is a peptide. The length of the peptide spacer may be, for example, 1-20 amino acids, such as 4-20 amino acids in length. For example, a suitable spacer may comprise a plurality of Gly residues, Ser residues or both, or be composed thereof. Optionally, at least one amino acid in an amino acid and / or spacer having a side chain containing a primary or secondary amine is a D-amino acid. Any of a variety of polymer backbones or scaffolds may be used. For example, the polymeric backbone or support can be a polyamide, a polysaccharide, a polyanhydride, a polyacrylamide, a polymethacrylate, a polypeptide, polyethylene oxide or a dendrimer. In one embodiment, the polymeric backbone or support comprises a variety of reactive functional groups, such as carboxylic acid, anhydride or succinimide groups. The polymeric backbone or support reacts with a cyclic peptide. In one embodiment, the cyclic peptide comprises any of a variety of different reactive functional groups (such as carboxylic acid, anhydride or succinimide groups), which reacts with the appropriate groups on the polymeric backbone. Alternatively, the monomeric units that can be combined to form a polymeric backbone or support are first reacted with the cyclic peptide, and the resulting monomer is polymerized. In another embodiment, the short chain is prepolymerized, functionalized, and then the mixture of short chains of different compositions is assembled into a longer polymer.

[0083] In some specific embodiments, the complement inhibitor comprises a cell-reactive functional group that reacts with a sulfhydryl (SH) group to form a covalent bond.

[0084] In some specific embodiments, the complement inhibitor comprises a cell-reactive functional group that reacts with an amine group to form a covalent bond.

[0085] The cyclic peptide in the present invention is used as an active ingredient of the complement inhibitor and is mainly used to inhibit the activation of C3 complement.

[0086] The present invention also provides a method for preparing the complement inhibitor of the present invention according to the amino acid sequence.

[0087] The cyclic peptides of the present invention can be prepared by various peptide synthesis methods, according to conventional peptide synthesis methods, via condensation of one or more amino acid residues. Other methods for synthesizing peptides or peptidomimetics by solid phase or liquid phase methods are well known to those skilled in the art. During peptide synthesis, the branched amino and carboxyl groups can be protected / deprotected as needed, using commonly known protecting groups. Modification using protecting groups of selective peptides and peptide derivatives is obvious to those skilled in the art.

[0088] Alternatively, certain peptides of the present invention can be produced by expression in an appropriate prokaryotic or eukaryotic system. For example, the DNA construct can be inserted into a plasmid vector suitable for expression in a bacterial cell (e.g., E. coli) or a yeast cell (e.g., Saccharomyces cerevisiae), or inserted into a baculovirus vector for expression in an insect cell or a viral vector for expression in a mammalian cell. This vector contains the regulatory elements required for DNA expression in a host cell, which are arranged in a manner that allows DNA expression in the host cell. Such regulatory elements required for expression include a promoter sequence, a transcription initiation sequence, and an optional enhancer sequence.

[0089] SEQ ID NO.1-6 and other peptides of similar design are suitable for production by in vitro or in vivo expression of nucleic acid molecules. DNA constructs encoding peptide concatemers can be introduced into in vivo expression systems, and the upper limit of concatemers depends on the expression system used. After the concatemers are produced, the cleavage between the C-terminal Asn and the following N-terminal G is achieved by exposing the polypeptide to hydrazine.

[0090] Peptides produced by expressing genes in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art.

[0091] In one embodiment, commercially available expression / secretion systems may be used whereby the recombinant peptide is expressed and thereafter secreted from the host cell for easy purification from the surrounding culture medium.

[0092] The above cyclic peptides can also be produced using a combination of gene expression and synthesis. For example, they can be produced by gene expression and then subjected to one or more post-translational synthesis processes, such as modifying the N- or C-terminus or cyclizing the molecule.

[0093] Secondly, the present invention provides the use of the above complement inhibitor in preparing medicines.

[0094] The complement inhibitor is used to treat complement-mediated disorders, specifically C3 complement-mediated disorders, and specifically inhibits the cleavage of C3 by C3 convertase into C3a and C3b.

[0095] The diseases targeted by the drug include, but are not limited to: any one or a combination of eye diseases, glomerulonephritis, systemic lupus erythematosus, pneumococcal infection, rash, inflammation, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), complement-mediated hemolysis, adult respiratory syndrome, heart disease, and complement-mediated injury.

[0096] The drug can also play a protective or therapeutic role in hemodialysis (Mastellos, DC et al., Compstatin: a C3-targeted complement inhibitor reaching its prime for bedside intervention.), renal transplantation (Mastellos, DC et al., Complement C3-targeted therapy: replacing long-held assertions with evidence-based discovery.) and hemorrhagic shock (van Griensven, M et al. Protective effects of the complement inhibitor compstatin Cp40 in hemorrhagic shock.), as disclosed in the prior art.

[0097] The inflammation includes, but is not limited to, acute inflammation or chronic inflammation. In some embodiments, the inflammation manifests as periodontitis. Periodontitis is a prevalent chronic oral disease caused by excessive inflammation caused by the formation of unhealthy biological microbial communities in the subgingival tooth area, and may lead to tooth loss, impaired chewing and nutritional status. The incidence of periodontitis is high, and early clinical and histological observations of human periodontitis show that periodontal inflammation and tissue destruction are associated with increased complement activity. Therefore, the drug of the present invention can be used to treat periodontitis by exerting the effect of complement inhibitors.

[0098] The complement-mediated injury includes, but is not limited to, any one or a combination of early tissue damage in infectious diseases, tumor rejection, ischemia / reperfusion (I / R) injury, and rejection after xenotransplantation.

[0099] The eye diseases include, but are not limited to, any one or a combination of macular degeneration, choroidal neovascularization, retinal neovascularization, ocular inflammation, and geographic lesions.

[0100] In some cases, the disease targeted by the drug can also be: atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); C3 glomerulonephritis (C3GN); C3 glomerulopathy; complement-mediated renal and glomerular inflammatory diseases; age-related macular degeneration (AMD); eye diseases characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation, or any combination thereof; paroxysmal nocturnal hemoglobinuria (PNH); cold agglutinin disease (CAD); warm antibody autoimmune hemolytic anemia (wAIHAs); sickle cell disease; transplant-associated thrombotic microangiopathy. Vascular disease; rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); autoimmune and autoinflammatory renal diseases; autoimmune myocarditis; multiple sclerosis; traumatic brain and spinal cord injury; cerebral, intestinal, and renal ischemia-reperfusion (IR) injury; spontaneous and recurrent miscarriage; antiphospholipid syndrome (APS); Parkinson's disease; Alzheimer's disease; neurodegenerative inflammatory diseases underpinned by abnormal synaptic remodeling, microglial activity, and cognitive decline; asthma; antinuclear cytoplasmic antigen-associated hypoimmune vasculitis (Wegener syndrome); non-lupus autoimmune skin diseases such as pemphigus, bullous pemphigoid, and epidermolysis bullosa; post-traumatic shock; cancer; gingivitis; and atherosclerosis.

[0101] The present invention also provides the use of the aforementioned complement inhibitor in treating the above diseases.

[0102] When the complement inhibitor is used for treatment, its dosage can be from milligram level to gram level, and the dosage can be adjusted for different indications. For example, when it is used for local indications, its dosage can be considered to be set to milligram level; when it is used for systemic indications, its dosage can be considered to be set to gram level. The local indications include the aforementioned eye diseases or periodontitis, etc.; the systemic indications include paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, etc. The above are only application methods that can be selected according to general treatment methods, and it does not mean that the complement inhibitors of the present invention can only be applied in the above manner.

[0103] "Local administration" or "local delivery" for the delivery of a composition or agent of the present invention refers to delivery that is independent of the transport of the composition or agent through the vascular system to its intended target tissue or site. The composition or agent can be delivered directly to its intended target tissue or site, or delivered to its vicinity, such as delivery in close proximity to the intended target tissue or site. For example, the composition can be delivered by injection or implantation of the composition or agent, or by injection or implantation of a device containing the composition or agent. After local administration near the target tissue or site, the composition or agent or one or more components thereof can diffuse to the intended target tissue or site. It should be understood that after local delivery is completed, a portion of the therapeutic agent (usually only a small portion of the administered dose) can enter the vascular system and be transported to another location, including back to its intended target tissue or site.

[0104] Furthermore, the present invention provides a medicament comprising the aforementioned complement inhibitor.

[0105] The drug includes any one or more of the aforementioned complement inhibitors or pharmaceutically acceptable salts thereof.

[0106] Concatemers or multimers of complement inhibitors or salts thereof may also be used in the present invention.

[0107] In some cases, the drug includes any one or more of SEQ ID NO.1-6 or their modifications.

[0108] The medicine may also include other pharmaceutically acceptable carriers or excipients.

[0109] The pharmaceutically acceptable carrier or excipient includes, but is not limited to, buffer, excipient, stabilizer, preservative, flavoring agent, flavoring agent, membrane permeation enhancer, enzyme inhibitor, pH regulator or other excipients that do not affect the activity of the inhibitor but may affect the drugability.

[0110] The dosage form of the drug includes but is not limited to injection and infusion. Those skilled in the art can select the drug dosage form according to the specific indication, which is not limited to the content disclosed in the present invention, and can also be a drug dosage form disclosed or undisclosed in the prior art.

[0111] The drug can be administered by subcutaneous injection or intravenous injection.

[0112] The administration method of the drug can be local injection, including but not limited to: injection through the blood, through the skin, through the eyes, and through the abdominal cavity.

[0113] The drug may be administered transdermally or orally, or other methods that can achieve therapeutic effects.

[0114] Preferably, the drug can be an ophthalmic preparation, preferably a vitreous injection or an eye drop preparation.

[0115] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by preparing its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkaline or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional salts or quaternary ammonium salts of the parent compound formed from inorganic or organic acids. Such conventional salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, and the like; and salts prepared from inorganic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. Certain acidic or basic compounds of the present invention may exist in the form of zwitterions. All forms of the compounds, including free acid, free base and zwitterions are included within the scope of the present invention.

[0116] As used herein, "treatment" refers to providing treatment, i.e., providing any type of medical or surgical treatment to the subject. The purpose of providing treatment may be to reverse, alleviate or inhibit the development of a disease, disorder or condition, prevent or reduce the likelihood of its occurrence, or to reverse, alleviate, inhibit or prevent one or more symptoms or phenomena of a disease, disorder or condition, prevent or reduce the likelihood of its occurrence. "Prevention" refers to preventing a disease, disorder, condition or its symptoms or phenomena from occurring in at least some individuals for at least a period of time. Treatment may include administering a medicament to a subject after one or more symptoms or phenomena of an indication condition (e.g., macular degeneration or diabetic retinopathy) occur, for purposes such as reversing, alleviating, reducing the severity of the condition and / or inhibiting or preventing the development of the condition, and / or reversing, alleviating, reducing the severity of one or more symptoms or manifestations of the condition and / or inhibiting one or more symptoms or phenomena of the condition. The composition of the present invention may be administered to a subject who has developed an ocular disease (e.g., exudative or non-exudative ARMD or diabetic retinopathy), or who has an increased risk of developing such a disease relative to a member of the general population. The compositions of the invention may be administered prophylactically, that is, before any symptoms or signs of a condition occur. Typically in such cases, the subject is at risk of developing the condition.

[0117] Beneficial effects of the present invention:

[0118] When the cyclic peptide provided by the present invention is used as the active ingredient of the complement inhibitor, it has good affinity and inhibitory activity to the C3 protein. When used as a drug, the complement inhibitor of the present invention has better pharmacokinetic properties such as in vivo stability and solubility, and improves compliance, and has a wide range of clinical applications.

[0119] In the present invention, the "in vivo stability" refers to the property of a drug to resist biological activities such as enzyme degradation in the body while maintaining its own structure and properties unchanged. It is generally expressed by the time that the radioactive drug in animal or human serum maintains its basic properties.

[0120] In the present invention, the "compliance" (Patient compliance / Treatment compliance) is also called compliance, compliance, and refers to the patient's behavior of taking treatment according to the doctor's prescription and in accordance with the doctor's instructions. It is customary to call the patient "cooperation"; otherwise it is called non-compliance. Compliance can be divided into three categories: full compliance, partial compliance (exceeding or underdosing, increasing or decreasing the number of medications, etc.) and complete non-compliance. In actual treatment, these three types of compliance each account for 1 / 3. The patient's compliance with a specific medication is the compliance of the specific medication.

[0121] In the present invention, the "pharmacokinetics" is referred to as pharmacokinetics, which mainly studies the dynamic changes of the body's disposition of drugs. It includes the absorption, distribution, biochemical conversion (or metabolism) and excretion of drugs in the body, especially the law of changes in blood drug concentration over time. Drug metabolism is related to human age, gender, individual differences and genetic factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 This is the mass spectrum of SEQ ID NO.1.

[0123] Figure 2 This is the mass spectrum of SEQ ID NO.2.

[0124] Figure 3 This is the mass spectrum of SEQ ID NO.3.

[0125] Figure 4 This is the mass spectrum of SEQ ID NO.4.

[0126] Figure 5 This is the mass spectrum of SEQ ID NO.5.

[0127] Figure 6 This is the mass spectrum of SEQ ID NO.6.

[0128] Figure 7 The HPLC spectrum of SEQ ID NO.1 is shown in FIG.

[0129] Figure 8 This is the HPLC spectrum of SEQ ID NO.2.

[0130] Fig. 9 This is the HPLC spectrum of SEQ ID NO.3.

[0131] Fig.10 This is the HPLC spectrum of SEQ ID NO.4.

[0132] Fig.11 This is the HPLC spectrum of SEQ ID NO.5.

[0133] Fig.12 This is the HPLC spectrum of SEQ ID NO.6.

[0134] Fig.13 Real-time binding curve of samples in BLI affinity experiment.

[0135] Fig.14 This is the fitting curve of sample 2 in the MST affinity experiment.

[0136] Fig.15 This is the fitting curve of the positive sample control in the MST affinity experiment. DETAILED DESCRIPTION

[0137] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0138] Example 1

[0139] In this example, the following cyclic peptides were prepared as active ingredients of complement inhibitors:

[0140] SEQ ID NO.1: Ac-ICV-W(CH3)-QD-2Nal-G-Aib-HRCT-NH2(CC);

[0141] SEQ ID NO.2: Ac-ICV-AzaTrp-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0142] SEQ ID NO.3: Ac-ICV-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0143] SEQ ID NO.4: Ac-IC-Abu-W(CH3)-QD-5F-Trp-G-Pip-HRCT-NH2(CC);

[0144] SEQ ID NO.5: Ac-IC-Abu-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0145] SEQ ID NO. 6: Ac-IC-Tyr-Bpa-QD-5F-Trp-G-Aib-HRCT-NH2(CC).

[0146] Cyclization is carried out through a disulfide bridge formed by Cys-2 and Cys-12. Cys-2 represents cysteine ​​(C) at position 2 in the general formula, and Cys-12 represents cysteine ​​(C) at position 12 in the general formula.

[0147] SEQ ID NO.1-6 of this example is synthesized by F-moc solid phase peptide synthesis, and purified by reverse phase high performance liquid chromatography (RP-HPLC) according to the known technology in the art. However, the present invention does not limit its specific preparation method. Any product obtained by the above sequence or modified by the preparation method disclosed or undisclosed in the prior art is within the scope of the present invention. Those skilled in the art are capable of optimizing the above active ingredients by conventional techniques and verifying the various activities of the optimized cyclic peptides, such as the activities shown in the experimental examples in some cases, but not limited to the data form presented in the experimental examples.

[0148] The mass spectra of SEQ ID NO.1-6 in this example are shown in Figure 1-6 , HPLC spectrum see Figure 7-12 .

[0149] Experimental Example 1 BLI affinity experiment

[0150] The BLI affinity experiment was used to verify the effects of the cyclic peptide of the present invention and the reference substance.

[0151] BLI refers to Bio-Layer Interferometry, the principle of which is that after visible light is emitted from the spectrometer, two reflection spectra are formed at the two interfaces of the optical film layer at the end of the sensor, and an interference spectrum is formed. Any change in the film thickness caused by molecular binding can be reflected by the displacement value of the interference spectrum.

[0152] Ni-NTA biosensor (Sino Biological Inc.) was loaded with His-tag recombinant human C3 protein, and then an association curve (ForteBio Octet) was generated with 7 cyclic peptides (SRQ ID NO. 1-6, numbered 1-6, the positive control group was Ac-ICVW(CH3)QDWGAHRCT-NH2 cyclic peptide disclosed in the prior art, numbered 7). Subsequently, a 90 s dissociation step was performed after a 60 s association phase to obtain the Kd value by fitting the curve.

[0153] The storage solution was dissolved in 15% acetonitrile, and the working solution was diluted in series using PBS at pH 8.0.

[0154] The results show that the Kd values ​​of the cyclic peptides shown in SEQ ID NO.1-6 of the present invention in the BLI affinity experiment are all less than 1000nM, the binding rate constants kon are all greater than 1000l / Ms, and the dissociation rate constants kdis are all less than 0.05l / Ms.

[0155] The real-time binding curves of each sample are shown in Fig.13 .

[0156] The results showed that all tested compounds had affinity for C3 protein, with the optimal activity being <100 nM.

[0157] Experimental Example 2 MST affinity experiment

[0158] This experiment uses MST affinity experiment to determine the Kd value of the binding of cyclic peptide to C3 protein. It was commissioned by Nanjing Zhongding Biotechnology Co., Ltd.

[0159] 1. Reagents and consumables:

[0160] 1) Fluorescent labeling kit: Monolith Protein Labeling Kit RED-NHS 2nd Generation Kit;

[0161] 2) C3 protein buffer: PBS;

[0162] 3) Cyclic peptide buffer: 0.5×PBS, 15% ACN;

[0163] 4) 20% Tween-20;

[0164] 5) MST NT115 standard capillary.

[0165] 2. Experimental equipment

[0166]

[0167] 3. Experimental methods and results

[0168] In a standard label-based MST experiment, up to 16 dilution gradients can be measured at one time. The concentration of the fluorescently labeled molecule (Target, labeling molecule, cyclic peptide) is kept constant, while the other molecule (ligand, non-labeling molecule, C3 protein) is diluted 2-fold.

[0169] 3.1 Ligand molecule dilution loading

[0170] 10 μL of gradient diluted non-labeled molecules (Ligand) and 10 μL of fixed concentration labeled molecules (Target) were mixed and allowed to react for 5 min. The mixed samples were loaded into glass capillaries and then analyzed by MST-NT.115 equipment.

[0171] 3.2Pretest and Binding Check

[0172] Before the experiment begins, a Pretest step is performed to detect the Target fluorescent labeling intensity, detect protein adsorption and aggregation, and determine the experimental parameters.

[0173] 3.3Binding Affrnity

[0174] After completing Binding Check, if there is no protein adsorption or aggregation and the signal-to-noise ratio meets the standard, start Binding Affinity.

[0175] Binding Affinity experimental parameters: The concentration of the three peptides was about 200nM, Buffer (0.5×

[0176] PBS, 15% ACN), protein concentration was 0.25 μM at maximum, and the dilution was performed in two-fold series.

[0177] If the small molecule is dissolved in an aqueous solution, the small molecule stock solution is directly diluted 16 times with a 2-fold concentration gradient. After dilution, each tube of solution is 10μL, and then 10μL of the diluted protein solution is added to mix, and then aspirated with a capillary and placed in the instrument for detection. In this experimental case, the small molecule (protein) is dissolved in PBS. After the gradient dilution is completed, 10μL of the diluted Target solution is added to each tube of solution, and the mixture is gently blown several times with a pipette, and then aspirated with a capillary and placed in the instrument for analysis.

[0178] 3.4 Experimental Results

[0179] Results Figure 14-15 . Fig.14 is the fitting curve of SEQ ID NO.2, Fig.15 is the fitting curve of the positive control.

[0180] The results show that the cyclic peptides shown in SEQ ID NO. 1-6 of the present invention all show affinity in the BLI affinity experiment, wherein the optimal Kd value is less than 100 nM. The larger the Kd value, the lower the affinity, and vice versa, the smaller the Kd value, the higher the affinity.

[0181] Experimental Example 3 Solubility Experiment

[0182] The synthesized SEQ ID NO. 1-6 products and the positive control were dissolved in PBS to measure the solubility.

[0183] The solubility experiment was carried out according to the following steps:

[0184] Weigh the peptide (5-10 mg) in a LoBind-Ependorf tube; add 20 μL PBS (pH 7.4), vortex the mixture and centrifuge at 16873 g for 2 minutes. Unless otherwise stated, if the corresponding peptide is not dissolved, add an additional 10 μL of PBS, then vortex and centrifuge until all precipitates disappear. The pH of the peptide solution is determined on a pH indicator strip or using a SevenExcellence pH meter (Mettler Toledo, Columbus, OH). The concentration of the final solution is determined based on the absorbance measured on a NanoDrop2000c spectrophotometer as described above.

[0185] Experimental Example 4 Determination of half inhibitory concentration value

[0186] Half inhibitory concentration values ​​of SEQ ID NO. 1-6 assessed by classical complement inhibition assay. The association constant (Ka), dissociation constant (Kd) and equilibrium dissociation constant (KD) were determined by surface plasmon resonance (SPR) experiments.

[0187] The complement inhibition experiment can refer to the following steps:

[0188] Evaluation was performed by an established enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with 50 μL of 1% ovalbumin in PBS for 2 hours at normal ambient temperature. The wells were blocked with 200 μL of 1% BSA in PBS for 1 hour and then coated with 50 μL of 1:1000 diluted α-ovalbumin polyclonal antibody in PBS for 1 hour. Between each step, the plate was washed three times with 200 μL of 0.05% Tween 20 in PBS (PBS-T). Serial dilutions of cyclic peptides prepared in veronal buffer (VBS; 5 mM veronal, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl2 and 0.5 mM MgCl2) were added to the 96-well plate, followed by the addition of human plasma (1:40 in VBS), and the mixture was incubated at room temperature for 15 minutes. After washing, complement activation (C3b / iC3b / C3d bound to immune complexes) was detected using an HRP-conjugated goat α-human C3 antibody (1:1000; MP Biomedicals). The reaction was performed using an HRP substrate (0.05% ABTS and 0.1% H2O2 dissolved in 0.1M sodium citrate, pH 4.2) and read at 405nm using a VICTOR multi-label plate reader (PerkinElmer, Waltham, MA). 100% complement activation was obtained in the absence of a C3 peptide inhibitor, and the absorbance data obtained at 405nm were converted to percentage of complement inhibition. The percentage of inhibition was plotted against the logarithm of the concentration, and the resulting data set was fitted to the equation "log (inhibitor) vs normalized response" using GraphPad Prism 5. The IC50 value was obtained from the fitting parameters of the average value obtained from three independent experiments. Surface Plasmon Resonance (SPR) Experiments The binding affinity and kinetic characteristics of the cyclic peptides were evaluated by SPR using a Biacore 3000 instrument (GE Healthcare, Piscataway Town, NJ).

[0189] Experimental Example 5 In vivo pharmacokinetic evaluation

[0190] SEQ ID NO. 1-6 and positive control are administered systemically or topically to experimental animals and samples are collected at different time points. Pharmacokinetic parameters are determined and can be evaluated by UPLC-ESI-MS analysis of the samples. C3 or other marker levels are determined for each animal. The total concentration of the original compound and the cleavage product is quantitatively determined.

[0191] In the present invention, the evaluation of pharmacokinetics is not limited to the forms presented in the experimental examples, and any evaluation method or index known to those skilled in the art can be used to evaluate the pharmacokinetic properties of the inhibitor provided by the present invention.

[0192] The above embodiments are presented only as some preferred implementation modes of the present invention and are not intended to limit the present invention. Conventional substitutions or equivalent substitutions made by those skilled in the art on the basis of the technical solutions disclosed in this application are all within the scope of protection of the present invention.

Claims

1. A complement inhibitor, characterized in that The active part includes a cyclic peptide or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide is as shown in the general formula I: General formula I: R1-IC-Xaa3-Xaa4-QD-Xaa7-G-Xaa9-HRCT-R2; wherein Xaa7 and Xaa9 are selected from uncommon amino acids, and Xaa3 and Xaa4 include at least one uncommon amino acid; R1 and R2 are modifying groups; Formula I is cyclized via a disulfide bridge formed between Cys-2 and Cys-12.

2. The complement inhibitor according to claim 1, characterized in that Xaa3 is selected from common amino acids or uncommon amino acids; Xaa4 is selected from uncommon amino acids; Xaa7 is selected from 2Nal or 5F-Trp; Xaa9 is selected from Aib or Pip.

3. The complement inhibitor according to claim 2, characterized in that Xaa3 is selected from any one of V, Y or Abu; Xaa4 is selected from any one of 2Nal, W(CH3), Bpa; Xaa7 is selected from any one of 2Nal or 5F-Trp; Xaa9 is selected from any one of Aib or Pip.

4. The complement inhibitor according to claim 1, characterized in that The active part is selected from any one or more of SEQ ID NO.1-6.

5. The complement inhibitor according to claim 1, characterized in that The product after optimization of general formula I as the active part; the optimization includes one or more of PEGylation, conjugation, homopolymer, heteropolymer, amino acid chirality optimization, hydrophilic / charged residue incorporation, increase of cell-penetrating peptide, and N-methylation in the chain.

6. The complement inhibitor according to claim 5, characterized in that The optimization occurs on R1 or R2 or the main chain amino acids; preferably, the optimization is the incorporation of hydrophilic / charged residues on R2, preferably the incorporation of any one or more K or R.

7. The complement inhibitor according to claim 5, characterized in that The PEGylation occurs at R1; the mid-chain N-methylation occurs at Xaa9.

8. A method for preparing the complement inhibitor according to any one of claims 1 to 7 based on the amino acid sequence.

9. Use of the complement inhibitor according to any one of claims 1 to 7 in the preparation of a medicament for treating complement-mediated disorders.

10. The use according to claim 9, characterized in that: The complement-mediated disorders include: any one or a combination of eye diseases, glomerulonephritis, systemic lupus erythematosus, pneumococcal infection, rash, inflammation, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, complement-mediated hemolysis, adult respiratory syndrome, heart disease, and complement-mediated injury; the inflammation includes periodontitis; the eye diseases include: any one or a combination of macular degeneration, choroidal neovascularization, retinal neovascularization, and geographic lesions; the drug is preferably an injection, preferably a local injection, preferably a subcutaneous injection or an intravenous injection; the drug is preferably an ophthalmic preparation; preferably a vitreous injection or an eye drop.

11. A drug comprising the complement inhibitor according to any one of claims 1 to 7 or a salt thereof or a tandem or multimer thereof.

12. The drug according to claim 11, characterized in that Including any one or more of SEQ ID NO.1-6.

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