Novel PACE4 inhibitor and application thereof in resisting osteoarthritis

Abstract

The invention discloses a novel PACE4 inhibitor and application of the novel PACE4 inhibitor in resisting osteoarthritis. The novel PACE4 inhibitory peptide designed and synthesized by the invention can effectively inhibit expression of extracellular PACE4 without influencing normal cells, and is stable in joints and unstable in plasma, so that the novel PACE4 inhibitory peptide has excellent safety and can be effectively used for preventing and treating OA.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel PACE4 inhibitor and application thereof 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 total joint replacement surgery is usually required. Although current treatments can relieve mild to moderate pain and inflammation associated with OA, they do not protect the cartilage from further damage and have not been shown to slow disease progression. Therefore, treatments that prevent or slow changes in joint structure and function would effectively meet a significant medical need that is currently unmet.

[0003] The main 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 a type of proteoglycan 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 essential for the function of its tissue. The 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 main enzymes that can hydrolyze chondrocyte-synthesized proteoglycans 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 the removal of the prodomain to be active. PACE4 has been identified as the proprotein convertase responsible for activating aggrecanase in OA, and therefore, it can become a new target for the development of OA therapeutic drugs.

[0004] Currently known roles of PACE4 in OA include: PACE4 was isolated from human OA cartilage culture; potential recombinant proADAMTS-4 and -5 were activated by recombinant PACE-4 in solution; addition of recombinant PACE-4 to human articular cartilage culture 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; siRNA-mediated inhibition of PACE4 expression blocked cartilage degradation in OA and normal human cartilage cultures stimulated with inflammatory cytokines.

[0005] With the aging of the population, the prevalence of OA is expected to continue to grow worldwide in the next few decades. The current treatment of OA focuses on controlling signs and symptoms, with non-steroidal anti-inflammatory drugs (such as COX-2 inhibitors) as the main therapeutic drugs. But in fact, in the treatment of OA, the development of an effective therapy that can significantly delay or even reverse the progression of OA disease is a major unmet clinical need in the current OA treatment field. At present, drugs that are believed to have cartilage-maintaining effects include diacerein, glucosamine, oxapril, and chondroitin sulfate. Therefore, in addition to these disease-modifying drugs, OA therapeutic drugs that can provide better pain relief, have lower side effects or a higher upper limit of efficacy still have great development prospects. At present, there are no PACE4 inhibitors that can be used for transarticular injection to treat OA with selective activity against PACE4, no cell permeability, stability in synovial fluid, stability in joints, instability in plasma, and water-soluble properties. Summary of the invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a novel PACE4 inhibitor and its use in anti-osteoarthritis. The present invention designs and synthesizes a novel PACE4 inhibitory peptide, which can effectively inhibit the expression of extracellular PACE4 without affecting normal cells, and is stable in joints and unstable in plasma, so that it has excellent safety and can be effectively used for the prevention and treatment of OA.

[0007] In the present invention, the term "peptide" refers to an oligomer of amino acids linked together by peptide bonds to a short polymer. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), the length of a peptide is generally about 50 amino acids or less (e.g., 50, 45, 40, 35, 30, 25, 20, 15, 10 or less, or a range therebetween (e.g., 10-30)). A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs and / or modified amino acids. A peptide may be a subsequence of a naturally occurring protein or a non-natural (artificial) sequence.

[0008] 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.

[0009] The first aspect of the present invention provides a polypeptide or a derivative thereof, wherein the polypeptide has a general formula as shown in formula (I):

[0010] Arg-AA2-AA3-Arg-AA4

[0011] Formula (I)

[0012] in,

[0013] AA2 is selected from Phe or Ala;

[0014] AA3 is selected from Arg or Lys;

[0015] AA4 is Thr or does not exist.

[0016] In some embodiments of the present invention, the polypeptide is a PACE4 inhibitory peptide, that is, a polypeptide having PACE4 expression inhibitory activity.

[0017] In some embodiments of the invention, the polypeptide is:

[0018] AA1-Arg-AA2-AA3-Arg-AA4;

[0019] The AA1 is selected from Arg, Pro or a combination thereof.

[0020] In some embodiments of the present invention, the AA1 is Arg, Pro, Arg-Pro or Pro-Arg.

[0021] In some embodiments of the present invention, the AA1 is Arg or Pro-Arg.

[0022] In some embodiments of the present invention, the amino acids in the polypeptide are in D configuration or L configuration.

[0023] In the present invention, by replacing some amino acid residues in the polypeptide master template (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 have better stability in the joints.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In some embodiments of the present invention, there are at least 3 basic amino acids in the polypeptide or its derivative.

[0028] 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:

[0029] (1) AA1 is Arg, AA3 is Arg or Lys;

[0030] (2) AA1 is Pro-Arg, AA3 is Arg or Lys; and

[0031] (3) AA1 is Arg-Pro, and AA3 is Arg or Lys.

[0032] 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.

[0033] In some embodiments of the present invention, there are modifications of intermediate residues in the polypeptide or its derivatives.

[0034] In some embodiments of the invention, the intermediate residue modification is a chemical modification.

[0035] In some embodiments of the present invention, the chemical modification includes but is not limited to 4-trifluoromethyl modification, 4-guanidinyl modification and 4-methylpiperidine modification.

[0036] 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.

[0037] In some embodiments of the invention, the middle residue is AA2.

[0038] In the present invention, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof are chemically and / or physically compatible with other ingredients in the formulation and physiologically compatible with the recipient.

[0039] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts and isomers.

[0040] In some specific 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.

[0041] In some specific embodiments of the present invention, the salt formed with an organic acid includes a salt formed with a basic or acidic amino acid.

[0042] In some specific 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 the following organic bases: trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N″-dibenzylethylenediamine, etc. Examples of salts formed with inorganic acids include salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Examples of salts formed with organic acids include salts formed with the following organic acids: 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, p-toluenesulfonic acid, etc. Examples of salts formed with basic amino acids include salts formed with the following basic amino acids: arginine, lysine, ornithine, etc. Examples of salts formed with acidic amino acids include salts formed with the following acidic amino acids: aspartic acid, glutamic acid, etc.

[0043] In some specific embodiments of the present invention, the isomers include optical isomers, stereoisomers, regioisomers, and geometric isomers.

[0044] In some specific embodiments of the present invention, the isomers are stereoisomers and tautomers.

[0045] 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.

[0046] 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.

[0047] In some specific embodiments of the present invention, the chemical modification includes at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, and ubiquitination.

[0048] In the present invention, the chemical modification is used to protect the polypeptide or its derivatives from being cleaved by peptidases.

[0049] In some specific embodiments of the present invention, the targeting moiety comprises at least one of a ligand, a receptor, and an antibody.

[0050] In some embodiments of the present invention, the fluorescent dye comprises FITC.

[0051] 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.

[0052] In some embodiments of the present invention, the peptide conjugate comprises:

[0053] Ac-RFKR-NH2

[0054] Ac-RFKRT-NH2

[0055] Ac-RFKR-dT-NH2

[0056] Ac-RRFKR-dT-NH2

[0057] Ac-dR-RFKR-dT-NH2

[0058] P-dR-RFKR-dT-NH2

[0059] dP-dR-RFKR-dT-NH2

[0060] Ac-RF(4-CF3)-KRT-NH2

[0061] Ac-RF(4-CF3)-KR-dT-NH2

[0062] Ac-RRF(4-CF3)-KR-dT-NH2

[0063] Ac-dR-RF(4-CF3)-KR-dT-NH2

[0064] P-dR-RF(4-CF3)-KR-dT-NH2

[0065] dP-dR-RF(4-CF3)-KR-dT-NH2

[0066] Ac-RA(4-Pip)-KRT-NH2

[0067] Ac-RA(4-Pip)-KR-dT-NH2

[0068] Ac-RRA(4-Pip)-KR-dT-NH2

[0069] Ac-dR-RA(4-Pip)-KR-dT-NH2

[0070] P-dR-RA(4-Pip)-KR-dT-NH2

[0071] dP-dR-RA(4-Pip)-KR-dT-NH2

[0072] Ac-RF(4-Guan)-KRT-NH2

[0073] Ac-RF(4-Guan)-KR-dT-NH2

[0074] Ac-RRF(4-Guan)-KR-dT-NH2

[0075] Ac-dR-RF(4-Guan)-KR-dT-NH2

[0076] P-dR-RF(4-Guan)-KR-dT-NH2

[0077] dP-dR-RF(4-Guan)-KR-dT-NH2.

[0078] The third aspect of the present invention provides a nucleic acid molecule comprising:

[0079] (1) A nucleic acid molecule encoding the polypeptide or its derivative or peptide conjugate described in the present invention;

[0080] (2) A sequence having at least 70% sequence identity with (1), and the sequence retains PACE4 inhibitory activity.

[0081] In some embodiments of the present invention, the nucleic acid molecule contains modified bases.

[0082] 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 signal transduction.

[0083] In some specific embodiments of the present invention, the sequence in (2) has at least 75%, 80%, 85%, or 90% sequence identity with (1).

[0084] In some specific embodiments of the present invention, the sequence described in (2) has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with (1).

[0085] A fourth aspect of the present invention provides a biological material containing or expressing any one of the polypeptide or its derivative, peptide conjugate and nucleic acid molecule of the present invention.

[0086] In some specific embodiments of the present invention, the biological material includes an expression cassette, a vector and a cell.

[0087] 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).

[0088] In some specific 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.

[0089] The fifth aspect of the present invention provides a method for preparing the polypeptide or its derivatives or peptide conjugates of the present invention, including obtaining them using the biomaterials described in the present invention, or obtaining them by solid phase synthesis.

[0090] In some specific embodiments of the present invention, based on the specific selection of the biological material, those skilled in the art can process it based on routine operations in the art. For example, when the biological material is a cell, the cell is cultured to produce the polypeptide. When the biological material is a vector, the vector is transferred into the cell by conventional techniques in the art, and after positive clones are screened, the polypeptide is produced using the positive clones.

[0091] In some specific embodiments of the present invention, the preparation method of some PACE4 inhibitory peptides is exemplified, which should not be understood as limiting the scope of the present invention unless otherwise stated or implied from the context. In addition, the features described in conjunction with various or specific embodiments should not be understood to be unsuitable for use in conjunction with other embodiments disclosed in the invention, unless such exclusivity is explicitly stated or implied from the context.

[0092] A sixth aspect of the present invention provides a PACE4 inhibitor, which contains the peptide conjugate of the present invention and a pharmaceutically acceptable excipient and / or carrier.

[0093] 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.

[0094] In the present invention, as pharmaceutically acceptable carriers, various organic or inorganic carrier substances commonly used as preparation raw materials can be used, without particular limitation, and can be formulated in the form of excipients, lubricants, binders, disintegrants in solid preparations; solvents, solubilizers, suspending agents, isotonic agents, buffers, analgesics, etc. in liquid preparations. In addition, preparation additives such as preservatives, antioxidants, stabilizers, colorants, sweeteners, etc. can also be used as needed.

[0095] The seventh aspect of the present invention provides a pharmaceutical composition comprising the peptide conjugate of the present invention and at least one second pharmaceutically active ingredient.

[0096] 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.

[0097] In some specific embodiments of the present invention, the second active pharmaceutical ingredient includes anti-inflammatory drugs, joint cartilage nutrition substances, analgesics and hormones.

[0098] 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.

[0099] 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.

[0100] In some specific embodiments of the present invention, the drug contains a therapeutically or preventively effective amount of the PACE4 inhibitory peptide or its derivative of the present invention.

[0101] In the present invention, the term "treatment" generally refers to treatment and therapy of humans or animals, wherein some desired therapeutic effects are achieved, for example, inhibition of disease progression, including decreased progression rate, stagnation of progression rate, regression of disease, improvement of disease and cure of disease. Treatment as a preventive measure (i.e., prevention) is also included.

[0102] In the present invention, 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" in the present invention includes administering the drug of the present invention to a patient who is expected to have a high risk of developing the disease due to some disease-related factors but has not yet developed the disease, or to a patient who has developed the disease but has no subjective symptoms. Or administering the drug of the present invention to a patient who is afraid of recurrence of the disease after treatment of the disease.

[0103] 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 brings a therapeutic effect to the subject, for example: in a subject to which the amount is administered, the symptoms or state of the disease are alleviated, reduced, or eliminated, or the development of the symptoms or state of the disease is delayed or inhibited, compared with a subject not administered the amount. The therapeutically effective amount can be appropriately determined by a doctor or professional according to the age, weight, sex, and severity of the symptoms of the subject.

[0104] 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.

[0105] In some specific embodiments of the present invention, the dosage form of the drug includes an injectable dosage form, such as an injection.

[0106] In some specific embodiments of the present invention, applicable subjects of the drug include subjects and patients.

[0107] In some embodiments of the present invention, the subject includes non-human animals and humans.

[0108] 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 patients or subjects of OA disease have no age or gender restrictions and can be children, adults, or the elderly, wherein children can be from newborns to 12 years old.

[0109] In some embodiments of the present invention, the anti-osteoarthritis includes treating and / or preventing osteoarthritis.

[0110] 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.

[0111] 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.

[0112] In the present invention, the PACE4 inhibitory peptide or its derivatives in the present invention have been confirmed to have preventive and therapeutic effects on osteoarthritis through in vitro and in vivo experiments. Paired amino acid convertase (PACE4) is expressed in degraded cartilage (extracellularly), thereby activating glycoproteinase ADAMTS-4 and inducing glycoprotein degradation, causing OA. The PACE4 inhibitory peptide or its derivatives in the present invention can effectively inhibit the expression of PACE4, thereby further effectively preventing and treating OA.

[0113] The beneficial effects of the present invention are:

[0114] The PACE4 inhibitory peptide or its derivatives in the present invention have an excellent inhibitory effect on PACE4, and its IC 50 <10nM. It showed IC for GAG released by osteoblast-like cells (BNC) stimulated by IL-1 50 The inhibitory activity was <3 μM and, when administered in an OA surgical model, it had a protective effect against cartilage degradation and reduced the expression of target biomarkers.

[0115] In addition, the PACE4 inhibitory peptide or its derivatives in the present invention cannot pass through cells. Therefore, it mainly achieves extracellular PACE4 inhibition, and does not require additional selective inhibition of other pro-proteases that mainly exist in cells, which simplifies the difficulty of drug design.

[0116] 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.

[0117] In addition, the PACE4 inhibitory peptide or its derivatives in the present invention have good human pharmacokinetic / pharmacodynamic characteristics, and are stable in synovial fluid and OA cartilage (half-life in cartilage is greater than 7 days), but unstable in plasma, so that the peptide parent and corresponding metabolites can be quickly cleared by the kidneys, thereby improving its safety. Moreover, at an effective dose, it has not been found to have irritation or toxic side effects when administered intra-articularly and subcutaneously (100 times the effective dose).

[0118] The PACE4 inhibitory peptide or its derivatives in the present invention provides a new direction and option for the development of OA treatment-related drugs, and has the potential to be used 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

[0119] Figure 1 This is the HPLC spectrum of Ac-RFKR-NH2.

[0120] Figure 2 This is the mass spectrum of Ac-RFKR-NH2.

[0121] Figure 3 This is the HPLC spectrum of Ac-dR-RFKR-dT-NH2.

[0122] Figure 4 This is the mass spectrum of Ac-dR-RFKR-dT-NH2.

[0123] Figure 5 This is the HPLC spectrum of Ac-dR-RF(4-CF3)-KR-dT-NH2.

[0124] Figure 6 This is the mass spectrum of Ac-dR-RF(4-CF3)-KR-dT-NH2.

[0125] Figure 7 The HPLC spectrum of Ac-dR-RA(4-Pip)-KR-dT-NH2.

[0126] Figure 8 This is the mass spectrum of Ac-dR-RA(4-Pip)-KR-dT-NH2.

[0127] Fig. 9The HPLC spectrum of Ac-dR-RF(4-Guan)-KR-dT-NH2.

[0128] Fig.10 This is the mass spectrum of Ac-dR-RF(4-Guan)-KR-dT-NH2.

[0129] Fig.11 These are representative results of the stability of the PACE4 inhibitory peptides in the examples of the present invention in synovial fluid and plasma.

[0130] Fig.12 This represents the absorption (binding) effect of the PACE4 inhibitory peptide in the above examples in articular cartilage.

[0131] Fig.13 The therapeutic effect of Ac-RFKR-NH2 on the cartilage phenotype of OA mice, wherein A is the Safranin O-fast green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0132] Fig.14 The therapeutic effect of Ac-dR-RFKR-dT-NH2 on the cartilage phenotype of OA mice, wherein A is the Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0133] Fig.15 The therapeutic effect of Ac-dR-RF(4-CF3)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein A is the Safranin O-fast green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0134] Fig.16 The therapeutic effect of Ac-dR-RA(4-Pip)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein A is the Safranin O-fast green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0135] Fig.17The therapeutic effect of Ac-dR-RF(4-Guan)-KR-dT-NH2 on the cartilage phenotype of OA mice, wherein A is the Safranin O-Fast Green staining of OA cartilage to evaluate the therapeutic effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0136] Fig.18 Figure 3 is the preventive effect of Ac-RFKR-NH2 on the cartilage phenotype of OA mice, where A is the Safranin O-fast green staining of OA cartilage to evaluate the preventive effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0137] Fig.19 Figure 3 is the preventive effect of Ac-dR-RF(4-Guan)-KR-dT-NH2 inhibitory peptide on the cartilage phenotype of OA mice, where A is the Safranin O-fast green staining of OA cartilage to evaluate the preventive effect of PACE4 inhibitory peptide; B is the analysis result of cartilage pathology score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0138] Fig. 20 The effect of Ac-dR-RA(4-Pip)-KR-dT-NH2 on the subchondral bone phenotype of OA mice, where A is the result of Safranin O-Fast Green staining; B is the analysis result of cartilage pathological score (OARSI) of different groups (n=8; *p<0.05, ***p<0.001).

[0139] Fig.21 This is the TRAP staining result of the PACE4 inhibitory peptide in the example of the present invention in the arthritis prevention test.

[0140] Fig. 22 The figures show the preventive and therapeutic effects of the PACE4 inhibitory peptide in the examples of the present invention on the cartilage phenotype of OA rats, wherein A is the Safranin O-Fast Green staining image of OA cartilage in the prevention experiment; B is the OARSI score analysis results of different groups (*p<0.05, ***p<0.001); C is the Safranin O-Fast Green staining image of OA cartilage in the treatment experiment; D is the OARSI score analysis results of different groups (***p<0.001).

[0141] Fig.23The expression regulation of the PACE4 inhibitory peptide in the example of the present invention on OA rat cartilage, wherein AE corresponds to the IHC staining and semi-quantitative analysis results of Col II (A), Mmp13 (B) and Adamts5 (C) as well as full-length Aggrecan and cleaved Aggrecan (i.e., NITEGE, E) in the articular cartilage of rats in each group on the 14th day after surgery (*p<0.05, **p<0.01, ***p<0.001).

[0142] Fig.24 : The therapeutic effect of the PACE4 inhibitory peptide in the example of the present invention on the subchondral bone phenotype of OA rats, wherein A is the TRAP staining of the subchondral bone of OA rats in different groups; B is the micro-CT reconstruction image of the subchondral bone of rats in different groups; CE are the bone density (C), bone density (BV / TV, D) and bone density (Tb.th, E) analysis diagrams of the subchondral bone of different groups, respectively.

[0143] Fig.25 The in vivo pharmacokinetic results of the PACE4 inhibitory peptide in the examples of the present invention in rat cartilage, wherein A is the in vivo imaging visualization of FITC-PX1 injected cartilage; B and C are the relative fluorescence intensity of FITC-PX1 in rat cartilage at different time points after IA injection; D and E are the measurement results of FITC-PX1 concentration in rat cartilage homogenate at different time points after IA injection.

[0144] Fig.26 Figure 3 Changes in gait parameters of the ipsilateral and contralateral hindlimbs of mice in different groups, including paw strength (A) and paw contact (imprint) area (B) obtained from CatWalk analysis.

[0145] Fig. 27 The left hind paw withdrawal thresholds of mice in different groups at different time points after surgery. DETAILED DESCRIPTION

[0146] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0147] In the present invention, "lowercase d" or "D-" in the peptide sequence represents a D-configuration amino acid. The brackets in the peptide sequence represent modification 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.

[0148] 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.

[0149] Example 1 Design and preparation of PACE4 inhibitory peptide

[0150] In this example, a design principle of PACE4 inhibition and a preparation method thereof are exemplified.

[0151] 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).

[0152] Table 1 PACE4 inhibitory peptides

[0153]

[0154]

[0155] The structural formulas of the synthesized PACE4 inhibitory peptides are as follows: SEQ ID NOs: 2 to 7:

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Example 2 In vitro inhibitory effect of PACE4 inhibitory peptide

[0162] The PACE4 inhibitory peptide obtained in the above embodiment was tested for the in vitro PACE4 inhibitory effect, and the specific steps were as follows: The PACE4 enzyme inhibition experiment was carried out in a solution of 20mM Bis-Tis pH 6.5, 1mM CaCl2 and 1.8mg / mL BSA. All tests were performed using pyroGlu-Arg-Val-Lys-Arg-methyl-coumaryl-7-amide (Bachem, CA) (100μM) as a substrate. The experiment was carried out at 37°C for 60min using a Gemini EM 96-well fluorescence spectrometer (Molecular Devices, CA) (Ex: 370nm, Em: 460nm). The PACE4 inhibitory peptide involved in this patent was added to the test solution at different concentrations to competitively inhibit the determination of the IC50 value of the polypeptide.

[0163] The results are shown in Table 2.

[0164] Table 2 IC of PACE4 inhibitory peptides against PACE4 50

[0165]

[0166]

[0167] The HPLC spectra corresponding to each sequence are as follows Figures 1 to 10 shown.

[0168] It can be seen from Table 1 that the PACE4 inhibitory peptide obtained in the embodiment of the present invention has a significant PACE4 inhibitory effect relative to the mother polypeptide, and in general, after chemical modification of AA2, its PACE4 inhibitory effect can be enhanced to a certain extent, in particular, after 4-Guan modification, 4-CF3 modification and 4-Pip modification of AA2, its effect is significantly better than that of the unmodified version.

[0169] Example 4 Stability of PACE4 Inhibitory Peptide

[0170] 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.

[0171] The representative results are shown in Fig.11As shown. It can be found that the PACE4 inhibitory peptide in the embodiment of the present invention shows strong stability in human synovial fluid, but is unstable in human plasma, which meets the design requirements for the PACE4 inhibitory peptide in the present invention. Through its stability in human synovial fluid and instability in plasma and rapid degradation, the safety of the drug can be effectively improved, so that it only acts in the joints and is ineffective in the whole body system. And this effect mainly depends on the protection of acetylation so that the N-terminus of the PACE4 inhibitory peptide in the present invention is not affected by the peptidases present in the synovial fluid. At the same time, the amidated group at the C-terminus protects its C-terminus from the peptidases in the synovial fluid, so that it is specifically protected from internal cleavage of various peptidases in the synovial fluid, so that it has sufficient time to integrate into the articular cartilage and exert its drug activity. For PACE4 inhibitory peptides in other systems such as plasma, because their structural design does not have anti-cutting activity under other system conditions, they will be rapidly degraded.

[0172] Example 5 Binding of PACE4 Inhibitory Peptides in Bone Joints

[0173] Another design requirement of the PACE4 inhibitory peptide in the embodiment of the present invention is: on the basis of ensuring its physicochemical properties, improve its retention time and binding effect in articular cartilage, so as to obtain a longer-lasting therapeutic effect with as few administrations as possible (such as only 4-6 administrations per year). Since about 10-15% of cartilage is cartilage proteoglycan, which contains a large amount of glycosaminoglycan, the articular cartilage is negatively charged.

[0174] Based on the above theory, the PACE4 inhibitory peptide in the above embodiment is designed to contain at least 3 basic amino acids, so that it can directly bind to cartilage and have inherent articular cartilage retention properties. By testing the PACE4 inhibitory peptide in the above embodiment in in vitro cartilage culture and in vivo and in vitro experiments injected into the knee joint of rats by injection, it was found that the PACE4 inhibitory peptide in the above embodiment can be effectively retained in cartilage for more than 2-3 weeks, and this long-term retention effect can be converted into the maintenance of therapeutic effect.

[0175] In addition, the binding ability of PACE4 inhibitory peptides to articular cartilage was also tested.

[0176] Take 15 mg of bovine nasal cartilage and culture it in a culture medium at 37 ° C for 24 hours according to conventional operations in the art. Then, after the PACE4 inhibitory peptide in the above embodiment is connected to a fluorescent group (5-fluorescein), it is placed in 200 μL of culture medium with the above-mentioned bovine nasal cartilage at a final concentration of 1000 μM, and the culture is continued and fresh culture medium is replaced every day. On the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th and 7th days of culture, take equal amounts of culture, add 1 mL of extract (containing guanidine hydrochloride with a final concentration of 4M, 50mM sodium acetate, pH 6.8) and extract the PACE4 inhibitory peptide absorbed in the cartilage overnight at 4 ° C. Finally, the fluorescent signal is detected at an excitation wavelength of 490nm and an emission wavelength of 520nm. And the polypeptide concentration is quantitatively calculated based on the standard curve to determine its binding ability with articular cartilage.

[0177] Example results are as follows Fig.12 shown.

[0178] Fig.12 The articular cartilage absorption effect 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) is exemplarily shown. It can be found that, unlike the uniform elimination of the control peptide, the PACE4 inhibitory peptide in the above examples has two different elimination stages, and the second stage based on the presence of the PACE4 inhibitory peptide in the above examples significantly slows down its elimination in cartilage, which may have a long drug half-life (t 1 / 2 ). Based on the test results of all PACE4 inhibitory peptides, it was found that the PACE4 inhibitory peptides in the above examples remained in the cartilage for >7 days, with a maximum binding capacity of about 800 μM, and were able to quickly bind to articular cartilage.

[0179] Example 6 Safety and therapeutic effects of PACE4 inhibitory peptides

[0180] The PACE4 inhibitory peptide in the present invention is required to be able to cause no local irritation or local toxicity after intra-articular injection at an effective intra-articular dose (20 μg / joint) in rats, and to cause no systemic side effects such as heart, liver or kidney when used for subcutaneous injection at 20 mg / kg.

[0181] At the same time, since the expression of PACE4 in OA cartilage is extracellular, but in normal cartilage it is strictly restricted to cells, the safety of the PACE4 inhibitory peptide in the present invention to normal cartilage can be effectively controlled by adding a design that limits the cell permeability of the PACE4 inhibitory peptide to avoid damage to normal cartilage.

[0182] In this example, a traumatic osteoarthritis model was established by performing a 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).

[0183] Specifically, in the therapeutic effect test, 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgical group) and an OA modeling + therapeutic administration group (surgical 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 killed 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.

[0184] Cartilage tissue was collected from each group as a sample. The collected cartilage tissue samples were decalcified according to the routine in the art, then embedded in paraffin and cut into 7 μm thick slices. Safranin O-fast green staining was performed, and OARSI scoring of cartilage degeneration was performed. At the same time, TRAP staining was also performed to determine the subchondral bone resorption of rats before and after MMT surgery and during PACE4 inhibitory peptide treatment.

[0185] Representative results of the control molecules and several PACE4 inhibitory peptides in the examples of the present invention are shown in the following example: Figures 13 to 17 shown.

[0186] 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.

[0187] Example 7 Preventive Effect of PACE4 Inhibitory Peptide

[0188] In this example, a traumatic osteoarthritis model was established by performing a medial meniscus tear (MMT) operation to verify the preventive effect of PACE4 inhibitory peptide on OA. In the preventive effect test, PACE4 inhibitory peptide was injected intra-articularly 3 days and 1 day before the modeling operation (i.e., administered 2 times in total). In the following examples, the dosage of PACE4 inhibitory peptide was 0.1 mg / kg animal body weight.

[0189] Specifically, in the preventive effect test, 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgical group) and an OA modeling + therapeutic administration group (surgical 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 killed 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.

[0190] 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.

[0191] Representative results of the control molecules and the PACE4 inhibitory peptides in the examples of the present invention are shown in the following example: Fig.18 and Fig.19 shown.

[0192] 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 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.

[0193] Example 8 Long-term therapeutic effect of PACE4 inhibitory peptide

[0194] At the same time, the effect of different administration times on the therapeutic effect was further tested. 24 SD rats were divided into 3 groups (including a control group, an OA modeling group (surgical group) and an OA modeling + treatment administration group (surgical 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 show 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 day after MMT surgery) to 4 times (intra-articular injection on the 3rd, 7th, 10th and 24th day after MMT surgery), and 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.

[0195] The results of using the PACE4 inhibitory peptide represented by Ac-dR-RA(4-Pip)-KR-dT-NH2 are shown as an example ( Fig. 20 ).

[0196] It can be found that through the above tests, it can be found that compared with the control group, the OARSI score was significantly increased after OA modeling and decreased in the PACE4 inhibitory peptide treatment group. Compared with the OA group, in the preventive test, the OARSI score of the intervention group of the peptide Ac-dR-RA(4-Pip)-KR-dT-NH2 was reduced by 20%. The OARSI score of the intervention group of the control peptide Ac-RFKR-NH2 was only reduced by 4%, showing no obvious 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 4-dose treatment test 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).

[0197] Tartrate-resistant acid phosphatase (TRAP) is a specific marker enzyme for osteoclasts. TRAP staining of bone tissue can help understand the bone resorption of osteoclasts. Fig.21 ) showed no significant differences between the groups in the arthritis prevention test, while in the treatment test, TRAP-positive deposits slightly increased 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 generation of osteoclasts in the subchondral bone on day 14 after MMT surgery.

[0198] Example 9 Further verification of the effect of PACE4 inhibitory peptide

[0199] In this embodiment, the inventors will demonstrate the therapeutic and preventive effects of the PACE4 inhibitory peptides in the embodiments of the present invention on cartilage degeneration from more dimensions. In the following experiments, only the relevant test results of Ac-dR-RA(4-Pip)-KR-dT-NH2 are shown as an example. It should be understood that it does not mean that the effect can only be obtained from Ac-dR-RA(4-Pip)-KR-dT-NH2. Other PACE4 inhibitory peptides in the embodiments of the present invention also have substantially equivalent effects.

[0200] (1) Histological and immunohistochemical verification:

[0201] 15 SD rats were divided into a sham operation group, an OA group, and an OA+PX1 group (i.e., a PACE4 inhibitory peptide administration group), with 5 rats in each group. The mice were subjected to surgical modeling of medial meniscus tear (MMT) according to the method in the above embodiment to construct the OA model required for the OA and OA+PX1 groups. Specifically, the specific steps of the MTT operation are as follows: after the experimental rats are anesthetized, the left knee is cut, the joint capsule is opened, and the medial meniscus is cut into two parts, front and back. It should be noted that no damage to the surrounding cartilage is caused. The wound is then sutured to complete the modeling. The rats after the modeling is completed are placed in a cage to monitor the recovery.

[0202] Among the above-mentioned experimental animals, some are used as materials for preventive experiments, and the other part is used as materials for therapeutic efficacy.

[0203] For the validation of the preventive effect, the experimental rats in this part were injected IA with PACE4 inhibitory peptide at a dose of 1 mM, 20 μL 1 day before MMT surgery, and the knee joints were collected on the 7th day after MMT surgery.

[0204] For the validation part of the therapeutic effect, the experimental rats in this part were injected IA with PACE4 inhibitory peptide at a dose of 1mM, 2020μL on the 3rd and 10th days after MMT surgery, and the knee joints were collected on the 14th day after MMT surgery.

[0205] The collected knee joints were decalcified, then embedded in paraffin, and further cut into 7 μm sections. Safranin O-fast green staining and OARSI scoring were then performed according to the method in the above example to evaluate cartilage degradation. At the same time, IHC staining based on Col II, Mmp13, aggrecan, and aggrecan degradation fragment (ie, NITEGE) was used to analyze the expression pattern of its specific markers. In addition, TRAP staining was performed to determine the subchondral bone resorption in rats before and after MMT surgery and during PACE4 inhibitory peptide treatment.

[0206] The results are as follows Figure 22-23 shown.

[0207] Taking the exemplary experimental results of .... as an example, it can be found that in the prevention and treatment effect verification test, the results of Safranin O-Fast Green staining showed that the administration of PACE4 inhibitory peptide can effectively improve the cartilage degradation and severe GAG ​​loss caused by surgery in the OA group. Compared with the control group, the OARSI score of the OA group was increased, while that of the drug administration group was decreased (such as Fig. 22 ). In addition, the OARSI score of the preventive group decreased by 24.8% compared with the OA group, while the OARSI score of the therapeutic group decreased by 39.8% compared with the OA group, indicating that the administration of PACE4 inhibitory peptides has preventive and therapeutic effects.

[0208] In addition, IHC staining results showed that MMT surgery could significantly reduce the expression level of cartilage formation marker Col II, while increasing the expression of metallopeptidase and aggregation proteinase (including Mmp13 and Adamts5) ( Fig.23 AC). Specifically, the expression of full-length aggrecan decreased in the OA group, while the expression of NITEGE increased. However, these changes were reversed by the administration of PACE4 inhibitory peptides. This suggests that the administration of PACE4 inhibitory peptides has an inhibitory effect on aggrecan cleavage during the development of OA ( Fig.23 D and 23E).

[0209] A set of experiments was reconstructed according to the above method to verify the long-term efficacy. The modeling steps were the same as above, and the administration method was modified as follows: starting from the first day after MMT surgery, PACE4 inhibitory peptide was injected IA at a dose of 1mM, 20μL, for a total of 3 injections, each with an interval of 7 days. Joint samples were collected on the 28th day after surgery.

[0210] X-ray and micro-CT were used to analyze the subchondral bone resorption of rats in each group. Meanwhile, paraffin sections were prepared according to the above method, and TRAP staining and expression level of osteoclast cell chemical markers were detected to determine the therapeutic effect of PACE4 inhibitory peptide on osteoclastogenesis activity of OA subchondral bone.

[0211] The results are as follows Fig.24 shown.

[0212] It was found that TRAP-positive deposition in the OA group was slightly increased compared with the control group, but this could be changed by PACE4 inhibitor treatment ( Fig.24 A). In addition, on the 14th day after MMT surgery, the PACE4 inhibitor group showed a slight inhibitory effect on osteoclastogenesis in the subchondral bone. On the 28th day after MMT surgery, it was found that it had a significant improvement effect on subchondral bone loss, which can be corresponded to the micro-CT analysis ( Fig.24 B), bone marrow density (BMD, Fig.24 C) The ratio of bone volume to total volume (BV / TV, Fig.24 D) and trabecular thickness (Tb.th, Fig.24 E) Semi-quantitative analysis results.

[0213] Other PACE4 inhibitory peptides also showed basically the same experimental results.

[0214] (2) In vivo pK test of PACE4 inhibitory peptides:

[0215] In the in vivo pK test in this example, two methods were used to quantify the in vivo concentration of the PACE4 inhibitory peptide at different time points.

[0216] The first is to verify by using a mechanical destruction method, specifically: taking dR-RA(4-Pip)-KR-dT-NH2 as an example, FITC-conjugated PACE4 inhibitory peptide (hereinafter referred to as: FITC-PX1) was injected into the experimental rats at a dose of 1mM, 20μL IA. Total articular cartilage was harvested at 0.5, 6, 12, 24, 48, 72, 96, 120, 144 and 168 hours. The total articular cartilage obtained was frozen with liquid nitrogen and ground with a pestle and mortar. The homogenate was then resuspended in PBS, and the total volume of articular cartilage in each group was adjusted to the same amount. The fluorescence of FITC-PX1 in the homogenate was then measured using a fluorescence spectrophotometer (PerkinElmer).

[0217] The second method was verified by in vivo imaging, specifically: taking dR-RA(4-Pip)-KR-dT-NH2 as an example, FITC-PX1 was injected into the experimental rats at a dose of 1mM, 20μL IA. Afterwards, the rats were monitored using an IVIS system (Tanon ABL X6 PRO) at 0.5, 6, 12, 24, 48, 72, 96, 120, 144 and 168 hours after injection to obtain continuous fluorescence images. Among them, the ideal filter conditions for FITC imaging were set to an excitation wavelength of 488nm and an emission wavelength of 525nm. Rats were imaged at all time points in a prone position. The grayscale photographic images and fluorescence images of each sample were analyzed and superimposed using Living Image software. The region of interest (ROI) was drawn on the signal and expressed in p / s / cm 2 / sr quantified the average radiation efficiency. The corresponding concentration inhibition curve was drawn and T was calculated using GraphPad Prism 10 1 / 2 In addition, the retention percentage at each time point was calculated to monitor the long-term retention properties of the PACE4 inhibitory peptides in articular cartilage.

[0218] Based on in vivo imaging ( Fig.25 AC) and mechanical destruction ( Fig.25 The pK parameter was calculated using the FITC-PX1 concentration results measured by DE. The results showed that the half-life (T 1 / 2 ). In addition, the amount of FITC-PX1 retained in the cartilage on the 7th day after intra-articular injection calculated by both measurement methods exceeded 4%. The final concentration of PACE4 inhibitory peptide in the cartilage on the 7th day after surgery can reach 0.45mM, indicating that PACE4 inhibitory peptide has a high retention potential in cartilage.

[0219] Other PACE4 inhibitory peptides also showed basically the same experimental results.

[0220] (3) Gait analysis after administration of PACE4 inhibitory peptide:

[0221] OA and OA+PX1 group modeling (a total of 20 C57BL / 6 mice, 10 mice in each group). In this embodiment, the modeling was performed by left hind limb joint medial meniscus (DMM) surgery. After surgery, IA was injected 4 times at a dose of 1mM and 5μL, each time at an interval of 7 days. Then behavioral tests were performed at 4, 8 and 12 weeks after surgery.

[0222] For behavioral testing, the CatWalk gait analysis system (Noldus Information Technology) was used to measure the gait parameters of mice under free movement. Specifically, each experimental mouse was placed individually on a walkway and allowed to walk freely, crossing from one side of the walkway to the other. When the paws of the experimental mice contacted the glass plate, the light was reflected downward, and the irradiated contact area was recorded using a high-speed color camera (the camera was located below the glass plate and connected to a computer running CatWalk software v7.1). The software automatically marks all areas containing pixels exceeding the set threshold (7 pixels), records and generates parameters including paw contact intensity and paw release area for further analysis. For each parameter, only the right hind paw (RH) and left hind paw (LH) of each animal were analyzed. The ratio of the hind paw parameters of the surgical side / healthy side (LH / RH) was calculated based on the parameters to reflect unilateral changes.

[0223] The results are as follows Fig.26 shown.

[0224] In gait analysis, the results showed that the hind paw strength ( Fig.26 A) and contact area ( Fig.26 B) percentage increased significantly, indicating that PACE4 inhibitory peptide effectively promoted the damage caused by surgery.

[0225] Other PACE4 inhibitory peptides also showed basically the same experimental results.

[0226] (4) Von Frey test:

[0227] According to the above example, the experimental mice were subjected to DMM surgery modeling and drug administration, and the pain level (i.e., paw withdrawal threshold) of the OA and OA+ treatment groups at 4, 8, and 12 weeks after surgery was measured by electronic Von Frey (IITC Electronic Von Frey Anesthesia Instrument 2390, USA). The specific measurement method is: the tip of the force sensor is vertically applied to the center of the foot pad of the experimental mouse through the bottom of the mesh, and the experimenter gradually increases the pressure to induce an obvious paw withdrawal reaction. The instrument will record the withdrawal threshold accordingly.

[0228] The results are as follows Fig. 27 shown.

[0229] It can be found that in the von Frey test, the paw withdrawal threshold decreased sharply after modeling, indicating that pain hypersensitivity increased during OA. In the OA+ treatment group, the increase in pain hypersensitivity was slower compared with the OA group ( Fig. 27 ), which suggests that PX1 has a significant analgesic effect in the development of OA.

[0230] Other PACE4 inhibitory peptides also showed basically the same experimental results.

[0231] In summary, the PACE4 inhibitory peptide in the embodiments of the present invention has a relatively strong therapeutic and preventive effect on cartilage degradation.

[0232] 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 equivalent replacement methods and are included in the protection scope 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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