Novel pace4 inhibitor and use thereof in Anti-osteoarthritis

Abstract

A novel PACE4 inhibitor and use thereof in anti-osteoarthritis. A novel PACE4 inhibitory peptide is designed and synthesized, which can effectively inhibit the expression of extracellular PACE4 without affecting normal cells, and is stable in joints and unstable in plasma, so that the PACE4 inhibitory peptide has excellent safety and can be effectively used in the prevention and treatment of OA.

Description

A novel PACE4 inhibitor and its application in treating osteoarthritis Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel PACE4 inhibitor and its application in treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) pathology includes degeneration of articular cartilage, as well as subchondral bone sclerosis and osteophyte formation, all of which lead to impaired joint function. These structural changes are often accompanied by pain, limited motion, and joint instability, and therefore require total joint replacement surgery. Although current treatments can relieve mild to moderate pain and inflammation associated with OA, they cannot protect the cartilage from further damage and have not been proven to slow disease progression. Therefore, treatments that prevent or slow changes in joint structure and function will effectively meet a major medical need that is currently unmet.

[0003] The primary cause of cartilage loss is due to the proteolytic degradation of chondrocyte-synthesized proteoglycans and type II collagen. Chondrocyte-synthesized proteoglycans and type II collagen are key components of the extracellular matrix of cartilage, accounting for 90% of its dry weight. Chondrocyte-synthesized proteoglycans are proteoglycans containing glycosaminoglycans that hydrate the collagen network, thereby providing compressibility and elasticity to the cartilage. Therefore, maintaining the chondrocyte-synthesized proteoglycan content in articular cartilage is crucial for the function of the tissue. Loss of chondrocyte-synthesized proteoglycans is an early and key event in the progression of cartilage destruction, which is mainly attributed to the proteolytic cleavage of the aggrecan core protein by aggrecanase. Two cartilage aggrecanases, ADAMTS-4 and ADAMTS-5, have been identified as the primary enzymes that hydrolyze proteoglycans synthesized by chondrocytes in OA (see Tortorella MD, Malfait AM. Will the Real Aggrecanase(s) Step Up: Evaluating the Criteria that Define Aggrecanase Activity in Osteoarthritis. Curr Pharm Biotechnol. 2008; 9: 16-23.). These metalloenzymes are synthesized as latent enzymes and require removal of the prodomain to activate their activity. PACE4 has been identified as the proprotein convertase responsible for activating aggrecanase in OA and, therefore, could become a new target for the development of OA therapeutics.

[0004] Currently known roles of PACE4 in OA include: PACE4 was isolated from human OA cartilage cultures; potential recombinant proADAMTS-4 and -5 were activated by recombinant PACE-4 in solution; addition of recombinant PACE-4 to human articular cartilage cultures triggered aggrecanase-mediated catabolism similar to that observed in OA; PACE4 protein was localized to areas of OA cartilage undergoing fibrosis / degradation; inhibition of PACE4 using a broad-spectrum inhibitor blocked cartilage degradation in OA cartilage explants and normal human cartilage explant cultures stimulated with inflammatory cytokines; and siRNA-mediated inhibition of PACE4 expression blocked cartilage degradation in inflammatory cytokine-stimulated OA and normal human cartilage cultures.

[0005] With the aging population, the global prevalence of OA is expected to continue to increase over the next few decades. Currently, the treatment of OA focuses on controlling signs and symptoms, with nonsteroidal anti-inflammatory drugs (such as COX-2 inhibitors) as the primary treatment. However, developing an effective therapy that can significantly delay or even reverse OA disease progression is a significant unmet clinical need in the field of OA treatment. Currently, drugs believed to have cartilage-maintaining effects include diacerein, glucosamine, oxapril, and chondroitin sulfate. Therefore, in addition to these disease-modifying drugs, there is great potential for the development of OA treatments that can provide better pain relief, lower side effects, or a higher upper limit of efficacy. Currently, there are no PACE4 inhibitors that are selective for PACE4, lack cell permeability, are stable in synovial fluid, stable in joints, unstable in plasma, and water-soluble, suitable for transarticular injection into the treatment of OA.

[0006] Summary of the Invention

[0007] The present invention aims to address at least one of the aforementioned technical problems existing in the prior art. To this end, the present invention provides a novel PACE4 inhibitor and its use in treating osteoarthritis. The present invention has designed and synthesized a novel PACE4 inhibitory peptide that effectively inhibits extracellular PACE4 expression without affecting normal cells. It is stable in joints but unstable in plasma, resulting in an excellent safety profile and effective use in the prevention and treatment of OA.

[0008] In the present invention, the term "peptide" refers to oligomers of amino acids linked together by peptide bonds to short polymers. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are typically about 50 amino acids or less in length (e.g., 50, 45, 40, 35, 30, 25, 20, 15, 10 or less, or a range therebetween (e.g., 10-30)). Peptides may comprise natural amino acids, non-natural amino acids, amino acid analogs and / or modified amino acids. Peptides may be subsequences of naturally occurring proteins or non-natural (artificial) sequences.

[0009] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxyl group, the N-terminal amino group, and the side chain functional groups has been reversibly or irreversibly chemically blocked, or otherwise modified to another functional group.

[0010] The first aspect of the present invention provides a polypeptide or a derivative thereof, wherein the polypeptide has the general formula shown in formula (I): Arg-AA2-AA3-Arg-AA4 Formula (I)

[0011] in,

[0012] AA2 is selected from Phe or Ala;

[0013] AA3 is selected from Arg or Lys;

[0014] AA4 is Thr or does not exist.

[0015] In some embodiments of the present invention, the polypeptide is a PACE4 inhibitory peptide, that is, a polypeptide having PACE4 expression inhibitory activity.

[0016] In some embodiments of the invention, the polypeptide is: AA1-Arg-AA2-AA3-Arg-AA4;

[0017] The AA1 is selected from Arg, Pro or a combination thereof.

[0018] In some embodiments of the present invention, the AA1 is Arg, Pro, Arg-Pro or Pro-Arg.

[0019] In some embodiments of the present invention, the AA1 is Arg or Pro-Arg.

[0020] In some embodiments of the present invention, the amino acids in the polypeptide are in the D configuration or the L configuration.

[0021] In the present invention, by replacing some amino acid residues in the polypeptide master (SEQ ID NO: 1) with different configurations, the inhibitory effect of the PACE4 inhibitory peptide on PACE4 can be significantly improved. At the same time, the change of some amino acid configurations can also improve the resistance of the PACE4 inhibitory peptide to peptidases, thereby making it more stable in joints.

[0022] In the present invention, the polypeptide has at least three chondrocyte-synthesized proteoglycan binding sites, and the optional residues for binding to chondrocyte-synthesized proteoglycan include AA1, Arg after AA1, AA3, and Arg after AA3.

[0023] In some embodiments of the present invention, the residue for binding to chondrocyte-synthesized proteoglycan is Arg after AA1, Arg after AA3, and at least one of AA1 and AA3.

[0024] In some embodiments of the present invention, the residues for binding to chondrocyte-synthesized proteoglycan are Arg at the second position after AA1, Arg after AA3, and AA1 and AA3.

[0025] In some embodiments of the present invention, there are at least three basic amino acids in the polypeptide or its derivative.

[0026] In some embodiments of the present invention, at least two basic amino acids are present in AA1 and AA3. In the present invention, the situation where at least two basic amino acids are present in AA1 and AA3 includes:

[0027] (1) AA1 is Arg, AA3 is Arg or Lys;

[0028] (2) AA1 is Pro-Arg, AA3 is Arg or Lys; and

[0029] (3) AA1 is Arg-Pro, and AA3 is Arg or Lys.

[0030] In some specific embodiments of the present invention, AA1 and AA3 are selected as follows: AA1 is Arg, and AA3 is Arg or Lys; or AA1 is Pro-Arg, and AA3 is Arg or Lys.

[0031] In some embodiments of the present invention, there are modifications of intermediate residues in the polypeptide or its derivatives.

[0032] In some embodiments of the present invention, the modification of the intermediate residue is a chemical modification.

[0033] In some embodiments of the present invention, the chemical modification includes but is not limited to 4-trifluoromethyl modification, 4-guanidino modification and 4-methylpiperidine modification.

[0034] In some embodiments of the present invention, the intermediate residue includes any amino acid residue between the C-terminus and the N-terminus of the amino acid sequence of the polypeptide.

[0035] In some embodiments of the invention, the middle residue is AA2.

[0036] In the present invention, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with other ingredients in the formulation and physiologically compatible with the recipient.

[0037] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts and isomers.

[0038] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, and a salt formed with an organic acid.

[0039] In some embodiments of the present invention, the salt formed with an organic acid includes a salt formed with a basic or acidic amino acid.

[0040] In some embodiments of the present invention, examples of the metal salt include: alkali metal salts, such as sodium salts, potassium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, barium salts, etc.; and aluminum salts. Examples of salts formed with organic bases include salts formed with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N″-dibenzylethylenediamine, and the like. Examples of salts formed with inorganic acids include salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Examples of salts formed with organic acids include salts formed with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts formed with basic amino acids include salts formed with arginine, lysine, and ornithine, and examples of salts formed with acidic amino acids include salts formed with aspartic acid and glutamic acid, and the like.

[0041] In some specific embodiments of the present invention, the isomers include optical isomers, stereoisomers, regioisomers, and geometric isomers.

[0042] In some embodiments of the present invention, the isomers are stereoisomers and tautomers.

[0043] The second aspect of the present invention provides a peptide conjugate, which comprises the polypeptide or its derivative described in the present invention and a modified portion; the modified portion is located at the N-terminus and / or C-terminus of the polypeptide or its derivative.

[0044] In some embodiments of the present invention, the modification moiety comprises at least one of a chemical modification, a targeting moiety, a fluorescent dye, and a protein tag.

[0045] In some embodiments of the present invention, the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, and ubiquitination.

[0046] In the present invention, the chemical modification is used to protect the polypeptide or its derivatives from being cleaved by peptidases.

[0047] In some embodiments of the present invention, the targeting moiety comprises at least one of a ligand, a receptor, and an antibody.

[0048] In some embodiments of the invention, the fluorescent dye comprises FITC.

[0049] In some specific embodiments of the present invention, the protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP, and biotin.

[0050] In some embodiments of the present invention, the peptide conjugate comprises: Ac-RFKR-NH2 Ac-RFKRT-NH2 Ac-RFKR-dT-NH2 Ac-RRFKR-dT-NH2 Ac-dR-RFKR-dT-NH2 P-dR-RFKR-dT-NH2 dP-dR-RFKR-dT-NH2 Ac-RF(4-CF3)-KRT-NH2 Ac-RF(4-CF3)-KR-dT-NH2 Ac-RRF(4-CF3)-KR-dT-NH2 Ac-dR-RF(4-CF3)-KR-dT-NH2 P-dR-RF(4-CF3)-KR-dT-NH2 dP-dR-RF(4-CF3)-KR-dT-NH2 Ac-RA(4-Pip)-KRT-NH2 Ac-RA(4-Pip)-KR-dT-NH2 Ac-RRA(4-Pip)-KR-dT-NH2 Ac-dR-RA(4-Pip)-KR-dT-NH2 P-dR-RA(4-Pip)-KR-dT-NH2 dP-dR-RA(4-Pip)-KR-dT-NH2 Ac-RF(4-Guan)-KRT-NH2 Ac-RF(4-Guan)-KR-dT-NH2 Ac-RRF(4-Guan)-KR-dT-NH2 Ac-dR-RF(4-Guan)-KR-dT-NH2 P-dR-RF(4-Guan)-KR-dT-NH2 dP-dR-RF(4-Guan)-KR-dT-NH2.

[0051] The third aspect of the present invention provides a nucleic acid molecule comprising:

[0052] (1) A nucleic acid molecule encoding the polypeptide or its derivative or peptide conjugate described in the present invention;

[0053] (2) A sequence having at least 70% sequence identity with (1), and said sequence retains PACE4 inhibitory activity.

[0054] In some embodiments of the present invention, the nucleic acid molecule contains modified bases.

[0055] In some embodiments of the present invention, the modification does not affect the normal function of the nucleic acid molecule, including but not limited to: storage and transmission of genetic information and cell signaling.

[0056] In some embodiments of the present invention, the sequence in (2) has at least 75%, 80%, 85%, or 90% sequence identity with (1).

[0057] In some embodiments of the present invention, the sequence of (2) has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with (1).

[0058] In a fourth aspect, the present invention provides a biological material containing or expressing any one of the polypeptide or derivatives thereof, peptide conjugates and nucleic acid molecules of the present invention.

[0059] In some embodiments of the present invention, the biological material includes an expression cassette, a vector, and cells.

[0060] In some specific embodiments of the present invention, the vector includes but is not limited to a plasmid vector, a phage vector, a cosmid vector, a YAC (yeast artificial chromosome), and a BAC (bacterial artificial chromosome).

[0061] In some embodiments of the present invention, the cells include but are not limited to prokaryotic cells and eukaryotic cells, including bacteria, fungi, insect cells, and animal cells.

[0062] The fifth aspect of the present invention provides a method for preparing the polypeptide or its derivative or peptide conjugate of the present invention, which comprises obtaining the polypeptide or its derivative or peptide conjugate using the biomaterial described in the present invention or by solid phase synthesis.

[0063] In some embodiments of the present invention, based on the specific selection of the biomaterial, those skilled in the art can perform processing based on routine procedures in the art. For example, when the biomaterial is a cell, the cell is cultured to produce the polypeptide. When the biomaterial is a vector, the vector is transferred into the cell using conventional techniques in the art, and positive clones are screened for and then used to produce the polypeptide.

[0064] In some specific embodiments of the present invention, methods for preparing some PACE4 inhibitory peptides are exemplified. Unless otherwise stated or implied from the context, they should not be understood as limiting the scope of the present invention. In addition, features described in conjunction with various or specific embodiments should not be understood as being unsuitable for use in conjunction with other embodiments disclosed in the invention, unless such exclusivity is explicitly stated or implied from the context.

[0065] A sixth aspect of the present invention provides a PACE4 inhibitor comprising the peptide conjugate of the present invention and a pharmaceutically acceptable excipient and / or carrier.

[0066] In the present invention, the term "pharmaceutically acceptable excipient and / or carrier" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to pH regulators, surfactants, adjuvants and ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0067] In the present invention, pharmaceutically acceptable carriers may be any of a variety of organic or inorganic carrier substances commonly used as pharmaceutical raw materials, without particular limitation. These carriers may be incorporated into solid formulations in the form of excipients, lubricants, binders, and disintegrants; and liquid formulations in the form of solvents, solubilizers, suspending agents, isotonicity agents, buffers, and analgesics. Furthermore, pharmaceutical additives such as preservatives, antioxidants, stabilizers, colorants, and sweeteners may also be used as needed.

[0068] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the peptide conjugate of the present invention and at least one second pharmaceutically active ingredient.

[0069] In some specific embodiments of the present invention, the second active pharmaceutical ingredient is a drug conventionally used in the art for the treatment or auxiliary treatment of OA.

[0070] In some embodiments of the present invention, the second active pharmaceutical ingredient includes anti-inflammatory drugs, joint cartilage nutrition substances, analgesics and hormones.

[0071] In some specific embodiments of the present invention, anti-inflammatory drugs include non-steroidal anti-inflammatory drugs, such as ibuprofen, celecoxib, loxoprofen sodium, etc.; joint cartilage nourishing substances include glucosamine hydrochloride, chondroitin sulfate, etc.; analgesics include meloxicam, diclofenac sodium, etc.; hormones include steroid hormones.

[0072] The eighth aspect of the present invention provides use of the peptide conjugate of the present invention in the preparation of an anti-osteoarthritis drug.

[0073] In some embodiments of the present invention, the drug contains a therapeutically or prophylactically effective amount of the PACE4 inhibitory peptide or its derivative of the present invention.

[0074] In the present invention, the term "treatment" generally refers to treatment and therapy of humans or animals, wherein some desired therapeutic effect is achieved, for example, inhibition of disease progression, including reduction in the rate of progression, stagnation of the rate of progression, regression of the disease, improvement of the disease and cure of the disease. Treatment as a preventive measure (i.e., prevention) is also included.

[0075] As used herein, the term "prevention" with respect to a disease condition in a mammal refers to preventing or delaying the onset of the disease or preventing the manifestation of clinical or subclinical symptoms thereof. "Prevention" herein encompasses administering the medicaments of the present invention to patients who are predicted to be at high risk of developing the disease due to disease-related factors but have not yet developed the disease, or to patients who have already developed the disease but have not yet experienced symptoms. Alternatively, the medicaments of the present invention may be administered to patients who fear recurrence of the disease after treatment.

[0076] The dosage of the PACE4 inhibitory peptide or its derivative for OA in the present invention is not particularly limited, as long as it is a therapeutically effective amount. In the present invention, the term "therapeutically effective amount" refers to an amount that produces a therapeutic effect in a subject. For example, in a subject administered with this amount, the symptoms or condition of the disease are alleviated, reduced, or eliminated, or the progression of the symptoms or condition of the disease is delayed or inhibited, compared to a subject not administered with this amount. The therapeutically effective amount can be appropriately determined by a physician or professional based on the subject's age, weight, sex, and severity of symptoms.

[0077] In some specific embodiments of the present invention, the dosage form of the drug includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, emulsion, suspension, syrup, and drops.

[0078] In some specific embodiments of the present invention, the dosage form of the drug includes an injectable dosage form, such as an injection.

[0079] In some embodiments of the present invention, the applicable subjects of the drug include subjects and patients.

[0080] In some embodiments of the present invention, the subject includes non-human animals and humans.

[0081] In some embodiments of the present invention, the non-human animals include research animals and companion animals such as mice, rats, primates, monkeys, great apes, chimpanzees, canines (e.g., dogs), and felines (e.g., cats). The human subjects or patients of OA disease are not limited in age or gender and can be children, adults, or the elderly. Children can range from newborns to 12 years old.

[0082] In some embodiments of the present invention, the anti-osteoarthritis effect includes treating and / or preventing osteoarthritis.

[0083] A ninth aspect of the present invention provides a method for treating and / or preventing osteoarthritis, comprising: administering the peptide conjugate of the present invention to a subject in need thereof.

[0084] In some specific embodiments of the present invention, the dosage of the PACE4 inhibitory peptide or its derivative is a preventive and / or therapeutically effective amount.

[0085] In the present invention, the PACE4 inhibitory peptides or their derivatives have been shown to have preventive and therapeutic effects on osteoarthritis through in vitro and in vivo experiments. Paired amino acid convertase (PACE4) is expressed extracellularly in degrading cartilage, activating the glycoprotein ADAMTS-4 and inducing glycoprotein degradation, leading to OA. The PACE4 inhibitory peptides or their derivatives can effectively inhibit PACE4 expression, thereby further effectively preventing and treating OA.

[0086] The beneficial effects of the present invention are:

[0087] The PACE4 inhibitory peptide or its derivatives in the present invention have excellent inhibitory effects on PACE4, and its IC 50 <10nM. It shows IC for GAG released by osteoblast-like cells (BNC) stimulated by IL-1 50 It exhibits inhibitory activity of <3 μM and, when administered in an OA surgical model, protects against cartilage degradation and reduces the expression of target biomarkers.

[0088] In addition, the PACE4 inhibitory peptide or its derivatives in the present invention cannot penetrate cells. Therefore, it mainly achieves extracellular PACE4 inhibition, without the need for additional selective inhibition of other proteases that mainly exist in cells, simplifying the difficulty of drug design.

[0089] Moreover, the PACE4 inhibitory peptide or its derivative in the present invention has good water solubility in buffer solution due to the presence of multiple positively charged amino acids, thereby allowing intra-articular administration with a maximum concentration of 100 μM.

[0090] Furthermore, the PACE4 inhibitory peptides or their derivatives of the present invention exhibit favorable human pharmacokinetic / pharmacodynamic characteristics. They are stable in synovial fluid and OA cartilage (with a half-life greater than 7 days in cartilage), but unstable in plasma, enabling rapid renal clearance of the peptide parent and corresponding metabolites, thereby enhancing their safety. Furthermore, at effective doses, no irritation or toxic side effects have been observed with intra-articular or subcutaneous administration (100 times the effective dose).

[0091] The PACE4 inhibitory peptide or its derivatives in the present invention provide new directions and options for the development of OA treatment-related drugs, and have the potential to serve as the first intra-articular OA therapeutic drug (DMOAD), using PACE4 as a therapeutic target to achieve the treatment and prevention of OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 is the HPLC spectrum of Ac-RFKR-NH2.

[0093] Figure 2 is the mass spectrum of Ac-RFKR-NH2.

[0094] FIG3 is the HPLC spectrum of Ac-dR-RFKR-dT-NH2.

[0095] Figure 4 is the mass spectrum of Ac-dR-RFKR-dT-NH2.

[0096] Figure 5 is the HPLC spectrum of Ac-dR-RF(4-CF3)-KR-dT-NH2.

[0097] Figure 6 is the mass spectrum of Ac-dR-RF(4-CF3)-KR-dT-NH2.

[0098] FIG7 is an HPLC spectrum of Ac-dR-RA(4-Pip)-KR-dT-NH2.

[0099] FIG8 is a mass spectrum of Ac-dR-RA(4-Pip)-KR-dT-NH2.

[0100] FIG9 is an HPLC spectrum of Ac-dR-RF(4-Guan)-KR-dT-NH2.

[0101] Figure 10 is the mass spectrum of Ac-dR-RF(4-Guan)-KR-dT-NH2.

[0102] FIG11A shows representative results of the stability of the PACE4 inhibitory peptide in synovial fluid according to an example of the present invention.

[0103] FIG11B shows representative results of the stability of the PACE4 inhibitory peptide in plasma according to an embodiment of the present invention.

[0104] FIG. 12 shows the absorption (binding) effect of the PACE4 inhibitory peptides in the above examples in articular cartilage.

[0105] Figures 13A-B show the therapeutic effect of Ac-RFKR-NH2 on the cartilage phenotype of OA mice, wherein Figure 13A shows Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; Figure 13B shows the analysis results of cartilage pathology scores (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0106] Figures 14A-B show the therapeutic effect of Ac-dR-RFKR-dT-NH2 on the cartilage phenotype of OA mice, wherein Figure 14A shows Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; Figure 14B shows the analysis results of cartilage pathology scores (OARSI) in different groups (n=8; *p<0.05, ***p<0.001).

[0107] Figures 15A-B show the therapeutic effect of Ac-dR-RF(4-CF3)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein Figure 15A shows Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; Figure 15B shows the analysis results of cartilage pathology scores (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0108] Figures 16A-B show the therapeutic effect of Ac-dR-RA(4-Pip)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein Figure 16A shows Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; Figure 16B shows the analysis results of cartilage pathology scores (OARSI) in different groups (n=8; *p<0.05, ***p<0.001).

[0109] Figures 17A-B show the therapeutic effect of Ac-dR-RF(4-Guan)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein Figure 17A shows Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; Figure 17B shows the analysis results of cartilage pathology scores (OARSI) in different groups (n=8; *p<0.05, ***p<0.001).

[0110] Figures 18A-B show the preventive effect of Ac-RFKR-NH2 on the cartilage phenotype of OA mice, wherein Figure 18A shows Safranin O-Fast Green staining of OA cartilage to evaluate the preventive effect of PACE4 inhibitory peptide; Figure 18B shows the analysis results of cartilage pathology scores (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0111] Figures 19A-B show the preventive effect of Ac-dR-RF(4-Guan)-KR-dT-NH2 inhibitory peptide on the cartilage phenotype of OA mice, wherein Figure 19A shows Safranin O-Fast Green staining of OA cartilage to evaluate the preventive effect of PACE4 inhibitory peptide; Figure 19B shows the analysis results of cartilage pathology scores (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0112] Figures 20A-B show the effects of Ac-dR-RA(4-Pip)-KR-dT-NH2 on the subchondral bone phenotype of OA mice, where Figure 20A shows the results of Safranin O-Fast Green staining; Figure 20B shows the results of cartilage pathology score (OARSI) analysis of different groups (n=8; *p<0.05, ***p<0.001).

[0113] FIG21A shows the TRAP staining results of the PACE4 inhibitory peptide in the example of the present invention in the arthritis prevention test (day 7).

[0114] FIG21B shows the TRAP staining results of the PACE4 inhibitory peptide in the example of the present invention in the arthritis prevention test (day 14). DETAILED DESCRIPTION

[0115] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0116] In the present invention, "lowercase d" or "D-" in the peptide sequence represents an amino acid with a D configuration. Brackets in the peptide sequence represent modifying groups. For example, F(4CF3) represents 4-trifluoromethyl-modified phenylalanine (Phe, F), i.e., (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propionic acid; F(4Guan) represents 4-guanidino-modified phenylalanine, i.e., (S)-2-amino-3-(4-guanidinophenyl)propionic acid. F(4-Pip) represents 4-methylpiperidinyl-modified phenylalanine, i.e., (S)-2-amino-3-((4-methylpiperidinyl)phenyl)propionic acid.

[0117] In the following examples, all polypeptides are synthesized using solid-phase synthesis technology. Of course, based on the polypeptide structural formulas shown in the following examples, those skilled in the art can also synthesize them using other methods in the art, including but not limited to the solid-phase synthesis technology used in the following examples.

[0118] Example 1 Design and Preparation of PACE4 Inhibitory Peptides

[0119] In this example, a design principle of a PACE4 inhibitor and a preparation method thereof are exemplified.

[0120] Ac-RFKR-NH2 (SEQ ID NO: 1) was synthesized in the solid state and used as a master to obtain the following PACE4 inhibitory peptides (as shown in Table 1).

[0121] Table 1 PACE4 inhibitory peptides

[0122] The structural formulas of the synthesized PACE4 inhibitory peptides are shown below:

[0123] SEQ ID NO: 2-7:

[0124] SEQ ID NO: 8-13:

[0125] SEQ ID NO: 14-19:

[0126] SEQ ID NO: 20-25:

[0127] Example 2 In vitro inhibitory effect of PACE4 inhibitory peptides

[0128] The PACE4 inhibitory peptides obtained in the above examples were tested for their in vitro PACE4 inhibitory effects. The specific steps were as follows: PACE4 enzyme inhibition experiments were conducted in a solution of 20 mM Bis-Tis pH 6.5, 1 mM CaCl2, and 1.8 mg / mL BSA. All assays used pyroGlu-Arg-Val-Lys-Arg-methyl-coumaryl-7-amide (Bachem, CA) (100 μM) as a substrate. The experiments were conducted at 37°C for 60 minutes using a Gemini EM 96-well fluorescence spectrometer (Molecular Devices, CA) (Ex: 370 nm, Em: 460 nm). The PACE4 inhibitory peptides described in this patent were added to the test solution at various concentrations to determine the IC50 value of the peptides for competitive inhibition.

[0129] The results are shown in Table 2.

[0130] Table 2 IC values ​​of PACE4 inhibitory peptides against PACE4 50

[0131] The HPLC spectra corresponding to each sequence are shown in Figures 1 to 10.

[0132] As can be seen from Table 1, the PACE4 inhibitory peptides obtained in the examples of the present invention have significant PACE4 inhibitory effects compared to the parent polypeptide. In general, after chemical modification of AA2, its PACE4 inhibitory effect can be enhanced to a certain extent. In particular, the effects of AA2 after 4-Guan modification, 4-CF3 modification, and 4-Pip modification are significantly better than those of the unmodified version.

[0133] Example 4 Stability of PACE4 Inhibitory Peptides

[0134] In this example, the PACE4 inhibitory peptide obtained above was co-incubated with human synovial fluid and plasma, respectively, and then the stability was tested by HPLC after 0, 0.5 and 1 h, respectively, to determine its stability in human synovial fluid and plasma.

[0135] Representative exemplary results are shown in Figures 11A and 11B. It can be seen that the PACE4 inhibitory peptides in the examples of the present invention exhibit strong stability in human synovial fluid but are unstable in human plasma, which meets the design requirements for the PACE4 inhibitory peptides of the present invention. By combining their stability in human synovial fluid with their rapid degradation in plasma, the drug's safety can be effectively improved, limiting its effect to the joints and excluding systemic inactivity. This effect is primarily due to the acetylation protection that protects the N-terminus of the PACE4 inhibitory peptides of the present invention from the effects of peptidases present in synovial fluid. Simultaneously, the amidated group at the C-terminus protects the C-terminus from the effects of peptidases in synovial fluid, thereby specifically protecting it from internal cleavage by various peptidases in synovial fluid. This allows it to integrate into articular cartilage and exert its pharmacological activity. In contrast, PACE4 inhibitory peptides in other systems, such as plasma, are rapidly degraded due to their structural design lacking resistance to cleavage under these conditions.

[0136] Example 5 Binding of PACE4 Inhibitory Peptides in Bone Joints

[0137] Another design requirement for the PACE4 inhibitory peptides in the embodiments of the present invention is to improve their retention time and binding effect in articular cartilage while ensuring their physicochemical properties, thereby achieving a longer-lasting therapeutic effect with as few dosing times as possible (e.g., only 4-6 dosings per year are required). Because approximately 10-15% of cartilage is cartilage proteoglycan, which contains a large amount of glycosaminoglycans, articular cartilage has a negative charge.

[0138] Based on this theory, the PACE4 inhibitory peptides in the above examples are designed to contain at least three basic amino acids, enabling them to directly bind to cartilage and possess inherent articular cartilage retention properties. Testing of the PACE4 inhibitory peptides in the above examples in cartilage cultures in vitro and in vivo via injection into rat knee joints revealed that the PACE4 inhibitory peptides in the above examples can effectively retain in cartilage for more than 2-3 weeks, and this prolonged retention effect can translate into sustained therapeutic efficacy.

[0139] In addition, the binding ability of PACE4 inhibitory peptides to articular cartilage was also tested.

[0140] 15 mg of bovine nasal cartilage was taken and cultured in a culture medium at 37 ° C for 24 hours according to conventional procedures in the art. The PACE4 inhibitory peptide in the above example was then linked to a fluorescent group (5-fluorescein) and placed in 200 μL of culture medium containing the above bovine nasal cartilage at a final concentration of 1000 μM. The culture was continued and fresh culture medium was replaced every day. On the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days of culture, equal amounts of culture were taken, and 1 mL of extract (containing guanidine hydrochloride at a final concentration of 4 M, 50 mM sodium acetate, pH 6.8) was added to extract the PACE4 inhibitory peptide absorbed by the cartilage overnight at 4 ° C. Finally, the fluorescence signal was detected at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The peptide concentration was quantitatively calculated based on the standard curve to determine its binding ability to articular cartilage.

[0141] Exemplary results are shown in FIG12 .

[0142] FIG12 exemplarily shows the articular cartilage absorption effects of two peptides (Fluorescein-dR-RA(4-CF3)-KR-dT-NH2 and Fluorescein-dR-RF(4-Guan)-KR-dT-NH2) representing the PACE4 inhibitory peptides in the above examples and a control peptide (Fluorescein-RFGG-dT). It can be found that, unlike the uniform elimination of the control peptide, the PACE4 inhibitory peptides in the above examples have two different elimination phases. The second phase based on the presence of the PACE4 inhibitory peptide in the above examples significantly slows its elimination in cartilage, which may be due to the long drug half-life (t) in cartilage. 1 / 2 ). Based on the test results of all PACE4 inhibitory peptides, it was found that the PACE4 inhibitory peptide in the above examples remained in the cartilage for >7 days, with a maximum binding capacity of approximately 800 μM, and was able to quickly bind to articular cartilage.

[0143] Example 6 Safety and therapeutic effects of PACE4 inhibitory peptides

[0144] The PACE4 inhibitory peptide of the present invention is required to be able to cause no local irritation or local toxicity after intra-articular injection in rats at an effective intra-articular dose (20 μg / joint), and to not cause any systemic side effects such as heart, liver or kidney when used for subcutaneous injection at 20 mg / kg.

[0145] At the same time, since PACE4 expression in OA cartilage is extracellular, while in normal cartilage it is strictly restricted to cells, the safety of the PACE4 inhibitory peptide in the present invention on normal cartilage can be effectively controlled by adding a design that limits the cell permeability of the PACE4 inhibitory peptide, thereby avoiding damage to normal cartilage.

[0146] In this example, a traumatic osteoarthritis model was established by performing medial meniscus tear (MMT) surgery to verify the therapeutic effect of PACE4 inhibitory peptides on OA. In the therapeutic effect test, injections were performed on the 3rd and 10th days after surgical modeling (i.e., a total of 2 doses).

[0147] Specifically, in the therapeutic effect test, 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgery group), and an OA modeling + therapeutic administration group (surgery group + drug intervention)), with 85 rats in each group. For the OA modeling + therapeutic administration group, intra-articular injection was performed on the 3rd and 10th days after MMT surgery. Rats in each group were sacrificed on the 14th day after modeling, and joint tissues were collected. Each PACE4 inhibitory peptide was tested separately to determine the therapeutic effect of each PACE4 inhibitory peptide. Among them, the polypeptide Ac-RFKR-NH2 (control molecule) was selected as a control example.

[0148] Cartilage tissue samples were collected from each group. The collected cartilage tissue samples were decalcified according to conventional methods in the art, then embedded in paraffin and cut into 7-μm-thick sections. Safranin O-Fast Green staining was performed, and cartilage degradation was assessed using OARSI scoring. TRAP staining was also performed to determine subchondral bone resorption in rats before and after MMT surgery and during treatment with a PACE4 inhibitory peptide.

[0149] Representative results of control molecules and several PACE4 inhibitory peptides in the examples of the present invention are shown in Figures 13A to 17B .

[0150] It can be found that in the therapeutic effect test, the results of Safranin O-Fast Green staining showed that the PACE4 inhibitory peptide in the embodiment of the present invention can effectively improve surgery-induced cartilage degeneration and severe GAG ​​(i.e., containing negatively charged carboxyl and sulfate groups, mainly released by osteoblast-like cells (BNC) under IL-1 stimulation) loss.

[0151] Example 7 Preventive Effect of PACE4 Inhibitory Peptide

[0152] In this example, a traumatic osteoarthritis model was established by performing medial meniscus tear (MMT) surgery to verify the preventive effect of PACE4 inhibitory peptides on OA. In the preventive effect test, the PACE4 inhibitory peptide was injected intra-articularly 3 days and 1 day before the modeling surgery (i.e., administered twice in total). In the following examples, the dosage of the PACE4 inhibitory peptide was 0.1 mg / kg animal body weight.

[0153] Specifically, in the preventive effect test, 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgery group), and an OA modeling + treatment administration group (surgery group + drug intervention)), with 8 rats in each group. For the OA modeling + preventive administration group, intra-articular injection was performed 3 days before and 1 day before MMT surgery. Rats in each group were sacrificed on the 7th day after modeling, and joint tissues were collected. Each PACE4 inhibitory peptide was tested separately to determine the preventive effect of each PACE4 inhibitory peptide. Among them, the polypeptide Ac-RFKR-NH2 (control molecule) was selected as a control example.

[0154] Safranin O-fast green staining, OARSI scoring, and TRAP staining were performed according to the methods in the above examples to determine the subchondral bone resorption in rats before and after MMT surgery and during PACE4 inhibitory peptide treatment.

[0155] Representative results of control molecules and PACE4 inhibitory peptides in the examples of the present invention are shown in Figures 18A-B and 19A-B .

[0156] It can be found that in the preventive effect test, the results of Safranin O-Fast Green staining showed that the PACE4 inhibitory peptide in the embodiment of the present invention can effectively prevent surgery-induced cartilage degradation and severe GAG ​​(i.e., containing negatively charged carboxyl and sulfate groups, mainly released by osteoblast-like cells (BNC) under IL-1 stimulation) loss.

[0157] Example 8 Long-term therapeutic effects of PACE4 inhibitory peptides

[0158] At the same time, the effect of different dosing times on the therapeutic effect was further tested. 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgery group), and an OA modeling + treatment administration group (surgery group + drug intervention)), with 8 rats in each group. Among them, in this embodiment, Ac-dR-RA (4-Pip) -KR-dT-NH2 is exemplified as a representative to demonstrate the similar effects of each PACE4 inhibitory peptide in the embodiment of the present invention. Among them, the number of administrations of Ac-dR-RA (4-Pip) -KR-dT-NH2 was changed from 2 times (intra-articular injection on the 3rd and 10th days after MMT surgery) to 4 times (intra-articular injection on the 3rd, 7th, 10th and 24th days after MMT surgery). Rats in each group were killed on the 31st day after modeling and joint tissues were collected. Safranin O-fast green staining, OARSI scoring, and TRAP staining were performed according to the methods in the above examples to determine the subchondral bone resorption in rats before and after MMT surgery and during PACE4 inhibitory peptide treatment.

[0159] The results of using a PACE4 inhibitory peptide represented by Ac-dR-RA(4-Pip)-KR-dT-NH2 are exemplified ( FIG. 20A-B ).

[0160] The above tests revealed that the OARSI score was significantly increased after OA modeling compared to the control group, but was decreased in the PACE4 inhibitory peptide treatment group. In the preventive test, the OARSI score in the Ac-dR-RA(4-Pip)-KR-dT-NH2 peptide intervention group decreased by 20% compared to the OA group. In contrast, the OARSI score in the control peptide Ac-RFKR-NH2 intervention group decreased by only 4%, demonstrating no significant preventive effect. In the therapeutic test, the OARSI scores of the intervention groups of Ac-dR-RFKR-dT-NH2, Ac-dR-RF(4-CF3)-KR-dT-NH2, Ac-dR-RA(4-Pip)-KR-dT-NH2 and Ac-dR-RF(4-Guan)-KR-dT-NH2 were reduced by 28%, 34.6%, 40.2% and 38.5 respectively, while the OARSI score of the intervention group of the control peptide Ac-RFKR-NH2 was only reduced by 10%, showing no significant therapeutic effect. At the same time, in the treatment test of four doses within 24 days, the OARSI score of the intervention group was reduced by 48.1%. The above results show that PACE4 inhibitory peptides have significant preventive and therapeutic effects (the therapeutic effect is relatively more significant).

[0161] Tartrate-resistant acid phosphatase (TRAP) is a specific marker enzyme for osteoclasts. TRAP staining of bone tissue can be used to understand the bone resorption of osteoclasts. The TRAP staining results (Figures 21A-B) showed that there was no significant difference between the different groups in the arthritis prevention test, while in the treatment test, TRAP-positive deposits increased slightly in the OA group and improved under Ac-dR-RA(4-Pip)-KR-dT-NH2 treatment. Therefore, it can be found that the use of Ac-dR-RA(4-Pip)-KR-dT-NH2 can slightly inhibit the formation of osteoclasts in the subchondral bone 14 days after MMT surgery.

[0162] In summary, the PACE4 inhibitory peptides in the embodiments of the present invention have relatively strong therapeutic and preventive effects on cartilage degradation.

[0163] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polypeptide or a derivative thereof, characterized in that: The polypeptide has the general formula shown in formula (I): Arg-AA2-AA3-Arg-AA4 Formula (I) in, AA2 is selected from Phe or Ala; AA3 is selected from Arg or Lys; AA4 is Thr or does not exist.

2. The polypeptide or derivative thereof according to claim 1, characterized in that: The polypeptide is: AA1-Arg-AA2-AA3-Arg-AA4; The AA1 is selected from Arg, Pro or a combination thereof.

3. The polypeptide or derivative thereof according to claim 1 or 2, characterized in that: The amino acids in the polypeptide or its derivatives are in D configuration or L configuration.

4. The polypeptide or derivative thereof according to claim 1 or 2, characterized in that: There are at least three basic amino acids in the polypeptide or its derivative.

5. The polypeptide or derivative thereof according to claim 1 or 2, characterized in that: There are modifications of intermediate residues in the polypeptide or its derivative.

6. The polypeptide or derivative thereof according to claim 5, characterized in that: The modification of the intermediate residue is a chemical modification, preferably including 4-trifluoromethyl modification, 4-guanidinyl modification and 4-methylpiperidine modification.

7. The polypeptide or derivative thereof according to claim 5 or 6, characterized in that: The middle residue is AA2.

8. The polypeptide or derivative thereof according to any one of claims 1 to 7, characterized in that: The derivatives include pharmaceutically acceptable salts and isomers.

9. The polypeptide or derivative thereof according to claim 8, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, and a salt formed with an organic acid.

10. The polypeptide or derivative thereof according to claim 8, characterized in that: The isomers include stereoisomers and tautomers.

11. A peptide conjugate, characterized in that The peptide conjugate comprises the polypeptide or its derivative according to any one of claims 1 to 10 and a modified portion; the modified portion is located at the N-terminus and / or C-terminus of the polypeptide or its derivative.

12. The peptide conjugate according to claim 11, characterized in that The modification moiety includes at least one of a chemical modification, a targeting moiety, a fluorescent dye, and a protein tag.

13. The peptide conjugate according to claim 12, characterized in that The chemical modification includes at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, and ubiquitination.

14. The peptide conjugate according to claim 11, characterized in that The peptide conjugate comprises: Ac-RFKR-NH2 Ac-RFKR-dT-NH2 Ac-RFKRT-NH2 Ac-RRFKR-NH2 Ac-dR-RFKR-dT-NH2 P-dR-RFKR-dT-NH2 dP-dR-RFKR-dT-NH2 Ac-RF(4-CF3)-KRT-NH2 Ac-RF(4-CF3)-KR-dT-NH2 Ac-dR-RF(4-CF3)-KR-dT-NH2 Ac-dR-RF(4-CF3)-KRT-NH2 P-dR-RF(4-CF3)-KR-dT-NH2 dP-dR-RF(4-CF3)-KR-dT-NH2 Ac-RA(4-Pip)-KR-dT-NH2 Ac-dR-RA(4-Pip)-KR-dT-NH2 Ac-RRA(4-Pip)-KR-dT-NH2 Ac-dR-RA(4-Pip)-KR-dT-NH2 P-dR-RA(4-Pip)-KR-dT-NH2 dP-dR-RA(4-Pip)-KR-dT-NH2 Ac-RF(4-Guan)-KR-dT-NH2 Ac-dR-RF(4-Guan)-KR-dT-NH2 Ac-RRF(4-Guan)-KR-dT-NH2 Ac-dR-RF(4-Guan)-KRT-NH2 P-dR-RF(4-Guan)-KR-dT-NH2 dP-dR-RF(4-Guan)-KR-dT-NH2.

15. A nucleic acid molecule, characterized in that include: (1) A nucleic acid molecule encoding the polynucleotide or its derivative according to any one of claims 1 to 10, or the peptide conjugate according to any one of claims 11 to 14; (2) A sequence having at least 70% sequence identity with (1), and the sequence retains PACE4 inhibitory activity.

16. The nucleic acid molecule according to claim 15, characterized in that The nucleic acid molecule contains modified bases.

17. A biological material containing or expressing the polypeptide or derivative thereof according to any one of claims 1 to 10, the peptide conjugate according to any one of claims 11 to 14, and any one of the nucleic acid molecules according to claim 15 or 16; the biological material comprises an expression cassette, a vector and a cell.

18. A method for preparing the polypeptide or derivative thereof according to any one of claims 1 to 10, or the peptide conjugate according to any one of claims 11 to 14, comprising obtaining the polypeptide using the biological material according to claim 17, or obtaining the polypeptide by solid phase synthesis.

19. A PACE4 inhibitor, characterized in that The PACE4 inhibitor contains the peptide conjugate according to any one of claims 11 to 14, and a pharmaceutically acceptable excipient and / or carrier.

20. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the peptide conjugate according to any one of claims 11 to 14 and at least one second pharmaceutically active ingredient.

21. The pharmaceutical composition according to claim 20, characterized in that The second active pharmaceutical ingredients include anti-inflammatory drugs, joint cartilage nutrition substances, analgesics and hormones.

22. Use of the polypeptide or derivative thereof according to any one of claims 1 to 10 and / or the peptide conjugate according to any one of claims 11 to 14 in the preparation of an anti-osteoarthritis drug.

23. The use according to claim 22, characterized in that The dosage form of the drug includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, emulsion, suspension, syrup and drops.

24. The use according to claim 22, characterized in that Applicable subjects of the drug include subjects and patients.

25. The use according to claim 24, characterized in that The subjects include non-human animals and humans.

26. The use according to claim 22, characterized in that The anti-osteoarthritis includes treating and / or preventing osteoarthritis.

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