Novel complement inhibitor, and preparation, use and product thereof

Abstract

Provided in the present invention are a novel complement inhibitor, and a preparation, the use and a product thereof, which belong to the field of molecular medicine. The portion having complement inhibitory activity comprises a cyclic peptide and a pharmaceutically acceptable salt thereof. The cyclic peptide is shown as general formula I: R1-ICXaa3-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. When the cyclic peptide provided in the present invention is used as an active ingredient of the complement inhibitor, the cyclic peptide has good affinity to a C3 protein, has good pharmacokinetic properties, and has good application prospects.

Description

Novel complement inhibitors and their preparation, application and products Technical Field

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

[0002] Complement is a group of globulins present in the blood, tissue fluid, and cell membranes of humans and animals that possess enzyme-like activity upon activation. The complement system is a crucial component of innate immunity, participating in immune regulation and immune-damaging responses. The complement system is composed of over 30 soluble and membrane-bound proteins, with activation pathways primarily consisting of the classical, alternative, and lectinactivation pathways. Complement exists in plasma in an inactive state but becomes biologically active upon activation, clearing immune complexes through lysis, opsonization, phagocytosis, and mediating inflammatory responses, demonstrating corresponding biological functions.

[0003] The three activation pathways of the complement system all revolve around the formation of C3 convertase and C5 convertase. Cleavage of C3 and C5 generates corresponding bioactive fragments, further amplifying the complement response. These fragments modify target surfaces and promote processes such as phagocytosis, inflammation, and immunomodulation. The process from contact with the activation initiator to the generation of C3 convertase (and subsequent cleavage of C3) can be considered the front-end reactions of these activation pathways. Although the front-end reactions of these activation pathways vary, they share a common terminal pathway: the generation of C5 convertase, which cleaves C5 to form the C5b fragment, which sequentially reacts with C6, C7, C8, and C9 to ultimately form the membrane attack complex (MAC), which exerts a cytolytic effect.

[0004] While the complement system plays a crucial role in human immunity, inappropriate or excessive complement activation is the root cause or contributing factor to many serious diseases. The discovery of complement inhibitors has provided promising research directions for the treatment of related diseases. Complement drugs have been approved for marketing in diseases such as paroxysmal nocturnal hemoglobinuria and geographic atrophy caused by age-related macular degeneration. Based on the three activation pathways of the complement system, it is known that inhibiting C3 can control the front-end reactions of the activation pathway. Therefore, the development of C3 complement inhibitors has great application prospects. C3, the most abundant complement protein in human serum, is composed of two chains, α and β. 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. Research progress 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 binds to both C3a and C3b and prevents the cleavage of native C3 by the C3 convertase. This cyclic peptide is composed of a polar portion, including a type I β-turn, and a non-polar portion, including a disulfide bond. Prior art has demonstrated that the four residues of the β-turn and the disulfide bond surrounding the hydrophobic cluster play a significant role in the inhibitory activity of compstatin. Based on the initially disclosed compstatin structure, prior art has continuously optimized it to improve its pharmaceutical properties. For example, the optimized compstatin analog, Ac-ICVW(CH3)QDWGAHRCT-NH2(CC), 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), the first marketed C3 complement inhibitor, is a synthetic cyclic peptide conjugated to a polyethylene glycol polymer that specifically binds to C3a and C3b. Empaveli's currently approved indications include paroxysmal nocturnal hemoglobinuria (PNH) and geographic atrophy caused by dry maculopathy.

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

[0008] Chinese patent application number CN201980030725.6 discloses a compstatin analog with C-terminal and / or N-terminal modifications, 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 for C3 and its fragments, but does not actually improve the properties of the cyclic peptide itself.

[0009] Some optimization approaches involve optimizing the amino acid profiles within the peptide, but there are many different approaches to amino acid optimization, and the results of any optimization approach are not definitive. Given the therapeutic potential of compstatin analogs in various diseases, further optimization of compstatin to achieve greater efficacy is crucial.

[0010] Summary of the Invention

[0011] In order to solve the above problems, the present invention provides a novel complement inhibitor and its preparation, application and product.

[0012] the term:

[0013] As used herein, "complement inhibitors," sometimes also referred to as compstatins or complement inhibitory analogs, are cyclic peptides that bind to the complement component C3 and inhibit complement activation. Compstatin inhibits the cleavage of C3 into C3a and C3b by the convertase enzyme. Because C3 is a central component of all three complement activation pathways, compstatin and its analogs can inhibit the activation of proteins that converge on all three pathways. The ability of compstatin and its analogs to inhibit the alternative pathway of complement activation may significantly contribute to their efficacy in certain ocular conditions described herein.

[0014] 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. The cyclic peptide can generally be formed by disulfide bonds, amide bonds, or other cyclic formation methods disclosed in the prior art. Multiple cyclic formation methods may exist in the same cyclic peptide.

[0015] As used herein, "polypeptide" refers to an amino acid polymer, optionally including one or more amino acid analogs. A protein is a molecule composed of one or more polypeptides. Peptides are relatively short polypeptides, typically ranging from about 2 to 60 amino acids in length. The terms "protein," "polypeptide," and "peptide" are used interchangeably. As used herein, a polypeptide may contain amino acids, such as amino acids naturally occurring in proteins, amino acids that do not naturally occur in proteins, and / or amino acid analogs that are not amino acids. As used herein, an amino acid "analog" may be a different amino acid that is structurally similar to an amino acid, or a compound other than an amino acid that is structurally similar to an amino acid. 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 a chemical entity (e.g., a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a conjugated linker), functionalization, or other modifications.

[0016] In the present invention, some abbreviations are interpreted as follows:

[0017] In the present invention, abbreviations not otherwise specified refer to the current commonly used abbreviations, specifically, the abbreviations of common amino acids.

[0018] In the present invention, the term "uncommon amino acids" encompasses all amino acid types other than the 20 commonly known amino acids. This includes both natural and unnatural amino acids. Natural amino acids may be discovered or undiscovered amino acids that occur naturally in nature. Unnatural amino acids may be artificially modified amino acids.

[0019] 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.

[0020] The present invention first provides a new complement inhibitor.

[0021] 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:

[0022] General formula I: R1-IC-Xaa3-Xaa4-QD-Xaa7-G-Xaa9-HRCT-R2(CC);

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The modification of the analogs can be methylation, acetylation, hydroxylation, carboxylation, phosphorylation, naphthylation, nitrogen atom hybridization, fluorination, acylation, alkoxylation, or halogenation.

[0027] Preferably, the tryptophan analog is selected from: 5-fluoro-DL-tryptophan, azatryptophan or N-methyltryptophan.

[0028] Preferably, when Xaa3 is selected from uncommon amino acids, it may be Abu;

[0029] Preferably, when Xaa3 is selected from common amino acids, it can be V or Y.

[0030] Preferably, when Xaa4 is selected from uncommon amino acids, it can be any one of 2Nal, W(CH3), and Bpa.

[0031] In some preferred conditions, the Xaa4 must be selected from uncommon amino acids.

[0032] Preferably, the Xaa7 is selected from 2Nal or 5F-Trp.

[0033] Preferably, the Xaa9 is selected from either Aib or Pip.

[0034] In some specific embodiments, Xaa3, Xaa4, Xaa7, and Xaa9 are each independently selected from the above preferred conditions, including but not limited to:

[0035] (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;

[0036] 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;

[0037] 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;

[0038] or in the cyclic peptide described in (4), Xaa3 is Abu, and Xaa7 is selected from either 2Nal or 5F-Trp;

[0039] or in the cyclic peptide described in (5), Xaa3 is Abu, and Xaa9 is selected from either Aib or Pip;

[0040] 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;

[0041] 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), 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] 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 groups, nitriles, 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, imines, 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, maleimides, sulfhydryls, and the like.

[0043] Preferably, the R1 may include one or more of hydrogen, acetyl, methoxy, PEG, and carboxyl.

[0044] Preferably, the R2 may include an amino group.

[0045] Preferably, the R1 or R2 includes an amino acid or a variant thereof.

[0046] In some embodiments, R1 or R2 independently comprises tyrosine or isoleucine.

[0047] In some embodiments, the R1 comprises isoleucine and the R2 comprises tyrosine.

[0048] Preferably, R1 and R2 form an amide bond to form a head-to-tail ring of the polypeptide.

[0049] In some embodiments, the complement inhibitory activity is partially selected from any one or more of SEQ ID NOs. 1-6:

[0050] SEQ ID NO.1: Ac-ICV-W(CH3)-QD-2Nal-G-Aib-HRCT-NH2(CC);

[0051] SEQ ID NO.2: Ac-ICV-AzaTrp-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0052] SEQ ID NO.3: Ac-ICV-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0053] SEQ ID NO.4: Ac-IC-Abu-W(CH3)-QD-5F-Trp-G-Pip-HRCT-NH2(CC);

[0054] SEQ ID NO.5: Ac-IC-Abu-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0055] SEQ ID NO. 6: Ac-IC-Y-Bpa-QD-5F-Trp-G-Aib-HRCT-NH2(CC).

[0056] Under some conditions, the general formula I has a structure similar to compstatin:

[0057] Cyclization is carried out via a disulfide bridge formed by Cys-2 and Cys-12. Cys-2 represents cysteine ​​(C) at position 2, and Cys-12 represents cysteine ​​(C) at position 12 in the general formula.

[0058] The complement inhibitor may directly include any one or more of the aforementioned cyclic peptides;

[0059] 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.

[0060] In the present invention, the term "side chain modification" may in some cases be referred to as "side chain modification" or "branch modification." As known to those skilled in the art, the termini of peptides are defined by default as free amino groups at the N-terminus and free carboxyl groups at the C-terminus. Peptide termini often require blocking, i.e., acetylation of the N-terminus and amidation of the C-terminus. This modification avoids the introduction of excess charge, enhances resistance to exonuclease action, and improves peptide stability.

[0061] 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.

[0062] The complement inhibitor may also be an optimized product of the aforementioned general formula I obtained by conventional techniques, without altering the activity of the cyclic peptide itself. Optimization may occur at R1 or R2, or at amino acids in the backbone. Examples include PEG optimization, conjugation, homopolymerization, heteropolymerization, amino acid chirality optimization, incorporation of hydrophilic / charged residues, addition of transmembrane peptides, and midchain N-methylation. Preferably, Xaa9 may include an N-methyl derivatization. The general formula I or its optimized form may be considered a compstatin analog.

[0063] In the present invention, "methylation" refers to the process of catalytically transferring a methyl group from an active methyl compound to another compound, which can form various methyl compounds, or chemically modify certain proteins to form methylated products. Specifically, the cyclic peptide provided by the present invention is chemically modified to form a methylated product.

[0064] In the present invention, "midchain N-methylation" refers to methylation occurring at any one or more amino acids in the chain. Specifically, a methyl group is attached to the -NH2 of any one or more amino acids in the chain to form an -NH-CH3 structure. Preferably, the midchain N-methylation occurs at any one amino acid in the chain. In some embodiments, the N-methylation occurs at amino acid position 9 of the cyclic peptide described herein.

[0065] In this invention, "amino acid chirality" refers to the configuration or conformation of an amino acid that is not identical to its mirror image and cannot overlap. Chirality is used to describe the relationship between an optically active molecule and its mirror image, which cannot overlap. All chiral molecules are optically active, and all compounds with optical activity are chiral molecules. Chiral molecules include asymmetric molecules without any symmetry factors and asymmetric molecules with a simple axis of symmetry but no other symmetry factors.

[0066] 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.

[0067] 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. They can enter the interior of the cell and interact with target molecules through mechanisms such as membrane perturbations and membrane flipping. Cell-penetrating peptides can play a role in drug transport. Cell-penetrating peptides are generally 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 the characteristics of good biostability, repeatable synthesis and change. In the present invention, the cell-penetrating peptide used as the complement inhibitor obtained by optimizing general formula I is not limited to its specific type. It can be composed of 3-30 amino acids or the composition of the number of amino acids 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 the cell-penetrating peptide TAT:H2N-YGRKKRRQRRR-OH.

[0068] The incorporation of hydrophilic / charged residues can be used to increase the solubility of cyclic peptides. Therefore, the complement inhibitors of Formula I can incorporate two or three hydrophilic / charged residues. These incorporation can occur at the C-terminus, N-terminus, or both. Specifically, in Formula I, these incorporation can occur at R1, R2, or both. Simultaneous incorporation refers to incorporation of hydrophilic / charged residues at both R1 and R2. In some cases, it can be assumed that peptide solubility increases with increasing the number of lysine residues, but this does not necessarily mean that such modifications have the same defined effect in all cases. Examples of residues that can be incorporated into Formula I include KK, KKK, KRK, and RRR. Adding two or three lysine residues can, in some cases, improve the solubility of cyclic peptides and, while maintaining inhibitory potency, enhance binding affinity, exhibit improved pharmacokinetic properties, expand the range of administration routes, reduce dosing frequency during chronic therapy, and improve drug compliance. This has been disclosed in some prior art, such as “Nadja B, Tchilabalo A, Resuello R, et al. New analogs of the complement C3 inhibitor compstatin with increased solubility and improved pharmacokinetic profile[J]. Journal of Medicinal Chemistry, 2018: acs.jmedchem.8b00560.”

[0069] Preferably, the hydrophilic / charged residue is incorporated into R2, specifically, before the amino group. More specifically, any one or more K or R residues are attached before the amino group. Alternatively, a single K or R residue may be attached; KK, RR, KR, or RK residues may be attached; or three amino acid residues may be attached, each of which is independently K or R residue, such as any of KKK, KKR, KRK, RKK, KRR, RKR, RRK, and RRR residues.

[0070] The complement inhibitor can be a cyclic peptide of the aforementioned general formula I, comprising a clearance-reducing moiety having a molecular weight of 1 kD to 100 kD. Specifically, the clearance-reducing moiety 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.

[0071] 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.

[0072] Preferably, the PEG can be branched PEG or 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.

[0073] 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.

[0074] Modifications, optionally linked to cell-reactive or targeting moieties, can be made by adding molecules (such as polyethylene glycol or similar molecules) to stabilize the compound, reduce its immunogenicity, prolong its in vivo lifespan, enhance or reduce its solubility, and / or enhance its resistance to degradation. Pegylation methods are well known in the art. Pegylation involves the chemical covalent coupling of activated PEG to a protein or polypeptide molecule. Complement inhibitors can be pegylated at the C-terminus, N-terminus, or both. Specifically, a polyethylene glycol molecule can be added to R1 or R2 of Formula I, or to both R1 and R2. Low-molecular-weight PEG chains can also be attached to the C-terminus or Lys residue of compstatin, as disclosed in the prior art, to develop novel analogs of the C3 inhibitor compstatin. Compared to the parent peptide, attachment of a 3 kD PEG can significantly improve solubility at physiological pH while maintaining inhibitory activity and exhibiting 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 the drug regulatory authorities of most countries / regions for intravenous, oral and dermal use in humans.

[0075] In some embodiments, it is well known to those skilled in the art that the solubility of a peptide can be enhanced by modifying the terminal region of a cyclic peptide, such as, but not limited to, PEG modification. Under certain conditions, those skilled in the art will recognize that in some cases the size of the PEG chain is directly related to the solubility of the compound, but this is not always the case, and other modifications may affect the solubility of the compound.

[0076] 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.

[0077] Preferably, the PEG-modified R2 of the general formula I 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 section on reduced clearance rate.

[0078] 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 moiety 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, by pegylation or conjugation to another moiety. 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 or multiple different substances.

[0079] In certain embodiments, a complement inhibitor is a multivalent compound comprising multiple cyclic peptides covalently or non-covalently linked to a polymeric backbone or scaffold. The linked cyclic peptides can be identical or different. In certain embodiments of the invention, the cyclic peptide comprises multiple instances or copies of a single compstatin analog moiety. 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 can be 1-10. In other embodiments of the invention, the number of cyclic peptides can be 10-20, 20-100, 100-1000, 1000-10,000, 10,000 to 50,000, 50,000 to 100,000, or 100,000 to 1,000,000.

[0080] The cyclic peptide can be directly linked to the polymeric scaffold, or can be linked via a linking moiety that links the cyclic peptide to the polymeric scaffold. The linking moiety can be linked to a single cyclic peptide and to the polymeric scaffold. Alternatively, the linking moiety can have multiple cyclic peptides linked thereto, such that the linking moiety links multiple cyclic peptides to the polymeric scaffold.

[0081] In certain embodiments, the cyclic peptide comprises an amino acid having a side chain containing a primary amine or secondary amine, such as a Lys residue. 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, for example, 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 the amino acid having a side chain containing a primary amine or secondary amine and / or the spacer 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 comprise 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 one of a variety of different reactive functional groups (such as carboxylic acid, anhydride or succinimide groups) that react with the appropriate groups on the polymeric backbone. Alternatively, the monomeric units that can be combined to form the polymeric backbone or support are first reacted with the cyclic peptide, and the resulting monomer is polymerized. In another embodiment, short chains are prepolymerized, functionalized, and then the mixture of short chains of different compositions is assembled into a longer polymer.

[0082] 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.

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

[0084] 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.

[0085] The present invention also provides a method for preparing the complement inhibitor of the present invention based on the amino acid sequence.

[0086] The cyclic peptides of the present invention can be prepared by various peptide synthesis methods, according to conventional peptide synthesis methods, through the 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 the peptide synthesis process, the branched amino and carboxyl groups can be protected / deprotected as needed using commonly known protecting groups. Modification using protecting groups of selected peptides and peptide derivatives will be apparent to those skilled in the art.

[0087] 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 bacterial cells (e.g., Escherichia coli) or yeast cells (e.g., Saccharomyces cerevisiae), or inserted into a baculovirus vector for expression in insect cells or a viral vector for expression in mammalian cells. This vector contains the regulatory elements required for DNA expression in the 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.

[0088] SEQ ID NOs. 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, with the upper limit of concatemer size depending on the expression system used. Following concatemer production, cleavage between the C-terminal Asn and the following N-terminal G is achieved by exposing the polypeptide to hydrazine.

[0089] Peptides produced by expressing genes in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art.

[0090] 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.

[0091] The 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.

[0092] Secondly, the present invention provides use of the aforementioned complement inhibitor in preparing medicines.

[0093] The complement inhibitor is used to treat complement-mediated disorders, specifically C3 complement-mediated disorders, and specifically inhibits the cleavage of C3 into C3a and C3b by C3 convertase.

[0094] 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.

[0095] 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.

[0096] 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 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 have shown 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 acting as a complement inhibitor.

[0097] 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.

[0098] 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.

[0099] In some cases, the disease for which the drug is directed 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 kidney 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 aberrant 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.

[0100] The present invention also provides the use of the aforementioned complement inhibitor in treating the above diseases.

[0101] When the complement inhibitor is used for treatment, its dosage can range from milligrams to grams, and the dosage can be adjusted for different indications. For example, when applied for local indications, its dosage can be considered to be set at the milligram level; when applied for systemic indications, its dosage can be considered to be set at the 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 selectable application methods based on general treatment methods, and do not mean that the complement inhibitors of the present invention can only be administered using the above methods.

[0102] With respect to the delivery of a composition or agent of the present invention, "local administration" or "local delivery" refers to delivery that is independent of 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 near it, for example, 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. Following 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 will be understood that after local delivery is complete, 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.

[0103] Furthermore, the present invention provides a drug comprising the aforementioned complement inhibitor.

[0104] The drug includes any one or more of the aforementioned complement inhibitors or pharmaceutically acceptable salts thereof.

[0105] Concatemers or multimers of complement inhibitors or salts thereof can also be used in the present invention.

[0106] In some cases, the drug includes any one or more of SEQ ID NO. 1-6 or modifications thereof.

[0107] The medicine may also include other pharmaceutically acceptable carriers or excipients.

[0108] The pharmaceutically acceptable carriers or excipients include, but are not limited to, buffers, excipients, stabilizers, preservatives, flavoring agents, flavoring agents, membrane permeability enhancers, enzyme inhibitors, pH regulators, or other excipients that do not affect the activity of the inhibitor but may affect the drugability.

[0109] The dosage form of the drug includes but is not limited to injections and infusions. 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.

[0110] The drug can be administered by subcutaneous injection or intravenous injection.

[0111] The drug can be administered by local injection, including but not limited to: injection through the blood, through the skin, through the eyes, and through the abdominal cavity.

[0112] The drug may be administered transdermally or orally, or other methods that can achieve therapeutic effects.

[0113] Preferably, the drug can be an ophthalmic preparation, preferably a vitreous injection or eye drop preparation.

[0114] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which the parent compound is modified by preparing an acid or base salt thereof. 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, sulfamic 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 zwitterionic form. All forms of the compounds, including free acids, free bases and zwitterions are included within the scope of the present invention.

[0115] 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 can be to reverse, alleviate or suppress the development of a disease, disorder or the patient's condition, prevent or reduce the possibility of its occurrence, or to reverse, alleviate, suppress or prevent one or more symptoms or phenomena of a disease, disorder or the patient's condition, prevent or reduce the possibility of its occurrence. "Prevention" refers to preventing a disease, disorder, the patient's 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 the subject after one or more symptoms or phenomena of an indicator patient's condition (such as macular degeneration or diabetic retinopathy) occur, for the purpose of, for example, reversing, alleviating, reducing the severity of the patient's condition and / or suppressing or preventing the development of the patient's condition, and / or reversing, alleviating, reducing the severity of one or more symptoms of the patient's condition or manifestation and / or suppressing one or more symptoms or phenomena of the patient's condition. The compositions of the present invention can be administered to a subject who has developed an ocular disease (such as exudative or non-exudative ARMD or diabetic retinopathy), or whose risk of developing such a disease increases relative to a member of the general population. The compositions of the invention can 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.

[0116] Beneficial effects of the present invention:

[0117] The cyclic peptide provided by the present invention, when used as the active ingredient of a complement inhibitor, exhibits good affinity and inhibitory activity against the C3 protein. When used as a pharmaceutical, the complement inhibitor of the present invention exhibits improved pharmacokinetic properties, such as in vivo stability and solubility, and has improved compliance, resulting in a wide range of clinical applications.

[0118] In the present invention, "in vivo stability" refers to the ability of a drug to resist biological activities such as enzymatic degradation in the body while maintaining its structure and properties. It is generally expressed by the time the radiopharmaceutical maintains its basic properties in animal or human serum.

[0119] In this application, "patient compliance" (also known as compliance or compliance) refers to a patient's behavior in following a doctor's prescribed treatment and adhering to the doctor's instructions. This is commonly referred to as patient "cooperation." The opposite is considered non-compliance. Compliance can be categorized into three types: complete compliance, partial compliance (overdosage or underdosage, increased or decreased frequency of medication use, etc.), and complete non-compliance. In actual treatment, these three types of compliance each account for one-third of the patient population. A patient's compliance with a specific medication is considered adherence to that medication.

[0120] In this context, "pharmacokinetics," also referred to as pharmacokinetics, primarily studies the dynamics of the body's disposition of drugs. This includes the processes of drug absorption, distribution, biochemical conversion (or metabolism), and excretion, particularly the temporal changes in blood drug concentrations. Drug metabolism is influenced by factors such as age, gender, individual differences, and genetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] FIG1 is a mass spectrum of SEQ ID NO.1.

[0122] Figure 2 is a mass spectrum of SEQ ID NO.2.

[0123] FIG3 is a mass spectrum of SEQ ID NO.3.

[0124] FIG4 is a mass spectrum of SEQ ID NO.4.

[0125] FIG5 is a mass spectrum of SEQ ID NO.5.

[0126] FIG6 is a mass spectrum of SEQ ID NO.6.

[0127] FIG7 is a HPLC spectrum of SEQ ID NO.1.

[0128] FIG8 is a HPLC spectrum of SEQ ID NO.2.

[0129] FIG9 is a HPLC spectrum of SEQ ID NO.3.

[0130] FIG10 is a HPLC spectrum of SEQ ID NO.4.

[0131] FIG11 is a HPLC spectrum of SEQ ID NO.5.

[0132] FIG12 is the HPLC spectrum of SEQ ID NO.6.

[0133] FIG13 shows the real-time binding curve of samples in the BLI affinity experiment.

[0134] FIG14 is the fitting curve of sample 2 in the MST affinity experiment.

[0135] FIG15 is a fitting curve of the positive sample control in the MST affinity experiment. DETAILED DESCRIPTION

[0136] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0137] Example 1

[0138] In this example, the following cyclic peptides were prepared as active ingredients of complement inhibitors:

[0139] SEQ ID NO.1: Ac-ICV-W(CH3)-QD-2Nal-G-Aib-HRCT-NH2(CC); z

[0140] SEQ ID NO.2: Ac-ICV-AzaTrp-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0141] SEQ ID NO.3: Ac-ICV-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0142] SEQ ID NO.4: Ac-IC-Abu-W(CH3)-QD-5F-Trp-G-Pip-HRCT-NH2(CC);

[0143] SEQ ID NO.5: Ac-IC-Abu-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC);

[0144] SEQ ID NO. 6: Ac-IC-Tyr-Bpa-QD-5F-Trp-G-Aib-HRCT-NH2(CC).

[0145] Cyclization is carried out via a disulfide bridge formed by Cys-2 and Cys-12. Cys-2 represents cysteine ​​(C) at position 2, and Cys-12 represents cysteine ​​(C) at position 12 in the general formula.

[0146] SEQ ID NO.1-6 of this embodiment is synthesized by F-moc solid phase peptide synthesis and purified by reverse phase high performance liquid chromatography (RP-HPLC) according to techniques known in the art. However, the present invention is not limited to its specific preparation method. Any product obtained by a preparation method disclosed or undisclosed in the prior art or modified to obtain the above sequence is within the scope of the present invention. Those skilled in the art are capable of performing conventional technical optimization on the above active ingredients and verifying the various activities of the optimized cyclic peptides, in some cases, as shown in the experimental examples, but not limited to the data form presented in the experimental examples.

[0147] The mass spectra of SEQ ID NO. 1-6 in this example are shown in Figures 1-6, and the HPLC spectra are shown in Figures 7-12.

[0148] Experimental Example 1 BLI affinity experiment

[0149] The BLI affinity experiment was used to verify the effects of the cyclic peptide of the present invention and the reference substance.

[0150] BLI stands for Bio-Layer Interferometry. The principle is that visible light emitted from a spectrometer forms two reflection spectra at the two interfaces of the optical film at the sensor's end, along with an interference spectrum. Any changes in film thickness caused by molecular binding are reflected by shifts in the interference spectrum.

[0151] A Ni-NTA biosensor (Sino Biological Inc.) was loaded with His-tag recombinant human C3 protein and then subjected to association curves (ForteBio Octet) using seven cyclic peptides (SRQ ID Nos. 1-6, numbered 1-6; the positive control was the Ac-ICVW(CH3)QDWGAHRCT-NH2 cyclic peptide disclosed in the prior art, numbered 7). A 60-second association phase was followed by a 90-second dissociation step to obtain Kd values ​​by curve fitting.

[0152] The storage solution was dissolved in 15% acetonitrile, and the working solution was diluted serially using PBS (pH 8.0).

[0153] The experimental results are shown in Table 1 below:

[0154] Table 1

[0155] The results showed that the Kd values ​​of the cyclic peptides represented by SEQ ID NO. 1-6 of the present invention in the BLI affinity experiment were all less than 1000 nM, the association rate constants kon were all greater than 1000 l / Ms, and the dissociation rate constants kdis were all less than 0.05 l / s.

[0156] The real-time binding curves of each sample are shown in FIG13 .

[0157] The results showed that all the tested compounds had affinity for C3 protein, with the optimal activity being <100 nM.

[0158] Experimental Example 2 MST affinity experiment

[0159] This experiment used an MST affinity assay to determine the Kd value of the cyclic peptide binding to the C3 protein. This was commissioned by Nanjing Zoonbio Biotechnology Co., Ltd.

[0160] 1. Reagents and consumables:

[0161] 1) Fluorescent labeling kit: Monolith Protein Labeling Kit RED-NHS2nd Generation Kit;

[0162] 2) C3 protein buffer: PBS;

[0163] 3) Cyclic peptide buffer: 0.5× PBS, 15% ACN;

[0164] 4) 20% Tween-20;

[0165] 5)MST NT115 standard capillary.

[0166] 2. Experimental equipment

[0167] 3. Experimental Methods and Results

[0168] In a standard label-based MST experiment, up to 16 dilution series can be measured at once. The concentration of the fluorescently labeled molecule (target, labeling molecule, cyclic peptide) is kept constant, while the other molecule (ligand, unlabeled molecule, C3 protein) is diluted in a two-fold series.

[0169] 3.1 Ligand molecule dilution loading

[0170] 10 μL of gradiently diluted non-labeled molecule (Ligand) and 10 μL of fixed concentration labeled molecule (Target) were mixed and allowed to react for 5 minutes. The mixed sample was loaded into a glass capillary and then analyzed by MST-NT.115 equipment.

[0171] 3.2 Pretest and Binding Check

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

[0173] 3.3 Binding 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 loading concentration of the three peptides was approximately 200 nM, the buffer was (0.5×PBS, 15% ACN), and the protein concentration was a maximum of 0.25 μM with 2-fold serial dilution.

[0176] If the small molecule is dissolved in aqueous solution, the stock solution is diluted 16 times in a 2-fold gradient, resulting in a 10 μL volume per tube. Then, 10 μL of the diluted protein solution is added, mixed thoroughly, and the solution is aspirated with a capillary tube before analysis. In this experimental case, the small molecule (protein) was dissolved in PBS. After the gradient dilution was completed, 10 μL of the diluted target solution was added to each tube. The solution was gently pipetted several times to mix thoroughly, then aspirated with a capillary tube before analysis.

[0177] 3.4 Experimental Results

[0178] The results are shown in Figures 14-15 and Table 2. Figure 14 is the fitting curve of SEQ ID NO. 2, and Figure 15 is the fitting curve of the positive control.

[0179] Table 2

[0180] The results showed that the cyclic peptides represented by SEQ ID NOs. 1-6 of the present invention all exhibited affinity in the BLI affinity assay, with the optimal Kd value being less than 100 nM. A larger Kd value indicates a lower affinity, while a smaller Kd value indicates a higher affinity.

[0181] Experimental Example 3 Solubility Experiment

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

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

[0184] Peptide (5-10 mg) was weighed in a LoBind-Ependorf tube; 20 μL of PBS (pH 7.4) was added, the mixture was vortexed and centrifuged at 16873 g for 2 minutes. Unless otherwise stated, if the corresponding peptide was not dissolved, an additional 10 μL of PBS was added, followed by vortexing and centrifugation until all precipitates disappeared. The pH of the peptide solution was determined on pH indicator strips or using a SevenExcellence pH meter (Mettler Toledo, Columbus, OH). The concentration of the final solution was determined based on the absorbance measured on a NanoDrop 2000c spectrophotometer as described above.

[0185] Experimental Example 4 Determination of half inhibitory concentration

[0186] The half inhibitory concentration values ​​of SEQ ID NO. 1-6 were evaluated by the 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] Complement inhibition experiments can be performed by following these 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 ambient temperature. The wells were blocked with 200 μL of 1% BSA in PBS for 1 hour and then coated with 50 μL of a 1:1000 diluted α-ovalbumin polyclonal antibody in PBS for 1 hour. Between each step, the plates were washed three times with 200 μL of 0.05% Tween 20 in PBS (PBS-T). Serial dilutions of the cyclic peptide 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 plates, 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 the immune complex) was detected using an HRP-conjugated goat α-human C3 antibody (1:1000; MP Biomedicals). Reactions were performed using an HRP substrate (0.05% ABTS and 0.1% H2O2 in 0.1 M sodium citrate, pH 4.2) and read at 405 nm using a VICTOR multilabel plate reader (PerkinElmer, Waltham, MA). 100% complement activation was obtained in the absence of a C3 peptide inhibitor, and the absorbance data obtained at 405 nm 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. IC50 values ​​were obtained from the fitting parameters of the average values ​​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] The experimental results are shown in Table 3 below:

[0190] Table 3

[0191] Experimental Example 5 In vivo pharmacokinetic evaluation

[0192] SEQ ID NOs. 1-6 and a positive control are administered systemically or topically to experimental animals, and samples are collected at various time points. Pharmacokinetic parameters are determined, which can be assessed by UPLC-ESI-MS analysis of the samples. C3 or other marker levels are measured in each animal. The total concentration of the original compound and cleavage products is quantified.

[0193] In the present invention, the evaluation of pharmacokinetics is not limited to the forms presented in the experimental examples, and any evaluation methods or indicators well known to those skilled in the art can be used to evaluate the pharmacokinetic properties of the inhibitors provided by the present invention.

[0194] The above embodiments are presented only as some preferred implementation methods 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 based on 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 portion 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, AzaTrp; 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 R1 is selected from one of hydrogen, acetyl, methoxy, PEG, and carboxyl, and R2 is selected from amino.

5. The complement inhibitor according to claim 4, characterized in that R1 is selected from acetyl, and R2 is selected from amino.

6. The complement inhibitor according to claim 1, characterized in that R1 and R2 are selected from amino acids or variants thereof.

7. The complement inhibitor according to claim 6, characterized in that R1 and R2 are each independently selected from tyrosine or isoleucine.

8. The complement inhibitor according to claim 7, characterized in that R1 is selected from isoleucine, and R2 is selected from tyrosine.

9. [Corrected 19.09.2024 in accordance with Rule 26] A complement inhibitor according to any one of claims 6 to 8, characterized in that R1 and R2 form an amide bond to form a head-to-tail ring of the polypeptide.

10. The complement inhibitor according to claim 1, characterized in that The active portion is selected from any one or more of SEQ ID NO.1-6: SEQ ID NO.1: Ac-ICV-W(CH3)-QD-2Nal-G-Aib-HRCT-NH2(CC); SEQ ID NO.2: Ac-ICV-AzaTrp-QD-5F-Trp-G-Aib-HRCT-NH2(CC); SEQ ID NO.3: Ac-ICV-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC); SEQ ID NO.4: Ac-IC-Abu-W(CH3)-QD-5F-Trp-G-Pip-HRCT-NH2(CC); SEQ ID NO.5: Ac-IC-Abu-2Nal-QD-5F-Trp-G-Aib-HRCT-NH2(CC); SEQ ID NO.6: Ac-IC-Y-Bpa-QD-5F-Trp-G-Aib-HRCT-NH2(CC).

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

12. The complement inhibitor according to claim 11, 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.

13. The complement inhibitor according to claim 11, characterized in that The PEGylation occurs at R1; the mid-chain N-methylation occurs at Xaa9.

14. A method for preparing the complement inhibitor according to any one of claims 1 to 13 based on the amino acid sequence.

15. Use of the complement inhibitor according to any one of claims 1 to 13 in the preparation of a medicament for treating complement-mediated disorders.

16. The use according to claim 15, 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.

17. A medicament comprising the complement inhibitor according to any one of claims 1 to 13, or a salt thereof, or a tandem or multimer thereof.

18. The drug according to claim 17, characterized in that Including any one or more of SEQ ID NO.1-6.

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