Drug for treating fracture and use thereof

Abstract

A drug for treating a fracture and a use thereof. Specifically, provided is a use of an E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or vascular endothelial growth factor-C (VEGF-C) in preparation of a drug for treating the fracture and / or promoting fracture healing. Provided is a pharmaceutical preparation for treating the fracture and / or promoting fracture healing. Experiments show that the E11 / gp38 / podoplanin / PDPN pathway inhibitor or the VEGF-C can significantly reduce swelling and pain at the end of fracture of a mouse, significantly improve the BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue, and remarkably improve the maximum stress, yield stress, and fracture energy of a fracture healing site, and has application prospects in the field of fracture treatment.

Description

Drugs for treating fractures and their applications Technical Field

[0001] The present invention relates to the field of medicine, in particular to a medicine for treating bone fractures and application thereof. Background Art

[0002] Traumatic fractures are common in orthopedics. Even with clinical treatment adhering to the three key principles of "good fixation, maintaining blood flow, and preventing infection," the incidence of delayed fracture healing and nonunion remains as high as 5-10%, with fractures of the femur and tibia and fibula being the most common. These injuries not only cause disability and pain to patients, but also place a heavy burden on families and society. Current Western medical approaches to promoting fracture repair primarily focus on enhancing bone conduction and induction, such as bone graft substitutes and bone growth factors. However, these approaches are expensive and their effectiveness is uncertain.

[0003] Therefore, finding new therapies to promote fracture healing is a research focus and hot topic in the field of orthopedics.

[0004] Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the following technical solution: Anti-PDPN is used in the preparation of a drug for preventing and treating fractures and / or promoting fracture healing.

[0006] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to reduce soft tissue swelling at the fracture ends of mice.

[0007] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to reduce the thermal pain threshold of mice.

[0008] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to increase BV / TV (bone volume fraction) and Tb.Th (trabecular bone thickness) of callus tissue.

[0009] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to increase the maximum stress and yield stress at the fracture healing site.

[0010] Furthermore, Anti-PDPN is used in the preparation of a drug for preventing and treating fractures and / or promoting fracture healing, comprising a therapeutically effective amount of Anti-PDPN or Anti-PDPN and a pharmaceutically acceptable excipient.

[0011] Anti-PDPN is used to prepare a pharmaceutical preparation for preventing and treating fractures and / or promoting fracture healing. The dosage form of the pharmaceutical composition is selected from the following group: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqueous solution, enteric-coated sustained-release preparation or injection.

[0012] Furthermore, the dosage form of the pharmaceutical composition is selected from the following group: oral dosage form, transdermal injection dosage form, intravenous injection dosage form, and intramuscular injection dosage form.

[0013] Regarding the beneficial effects, the present invention discloses the use of an anti-podoplanin (anti-PDPN, anti-specific recognition human podoplanin) neutralizing antibody in the preparation of a drug for preventing and treating fractures and / or promoting fracture healing. Also disclosed is a pharmaceutical preparation for preventing and treating fractures and / or promoting fracture healing. Experimental results show that anti-podoplanin neutralizing antibodies can significantly reduce swelling and pain at the fracture ends of mice, significantly increase the BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue, and significantly increase the maximum stress, yield stress and fracture energy at the fracture healing site. Therefore, anti-podoplanin neutralizing antibodies provide a new approach for fracture prevention and treatment.

[0014] Another object of the present invention is to provide the following technical solution: VEGF-C is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing.

[0015] Furthermore, VEGF-C is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to reduce soft tissue swelling at the fracture ends of mice.

[0016] Furthermore, VEGF-C is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to reduce the thermal pain threshold of mice.

[0017] Furthermore, VEGF-C is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, for increasing BV / TV (bone volume fraction) and Tb.N (trabecular thickness) of callus tissue and significantly reducing Tb.Sp (trabecular separation).

[0018] Furthermore, VEGF-C is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, and is used to increase the maximum stress, yield stress and fracture energy at the fracture healing site.

[0019] Furthermore, VEGF-C is used in the preparation of a drug for preventing and treating bone fractures and / or promoting fracture healing, comprising a therapeutically effective amount of VEGF-C or VEGF-C and a pharmaceutically acceptable excipient.

[0020] VEGF-C is used in the preparation of a pharmaceutical preparation for preventing and treating fractures and / or promoting fracture healing. The dosage form of the pharmaceutical composition is selected from the following group: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqueous solution, enteric-coated sustained-release preparation or injection.

[0021] Furthermore, the dosage form of the pharmaceutical composition is selected from the following group: oral dosage form, transdermal injection dosage form, intravenous injection dosage form, and intramuscular injection dosage form.

[0022] Regarding beneficial effects, the present invention discloses the use of vascular endothelial growth factor C (VEGF-C) in the preparation of a drug for preventing and treating fractures and / or promoting fracture healing. Also disclosed are pharmaceutical preparations for preventing and treating fractures and / or promoting fracture healing. Experimental results show that VEGF-C can significantly reduce swelling and pain at the fracture ends of mice, significantly increase the BV / TV (bone volume fraction) and Tb.N (trabecular thickness) of callus tissue and significantly reduce Tb.Sp (trabecular separation), and significantly increase the maximum stress, yield stress, and fracture energy at the fracture healing site. Therefore, VEGF-C provides a new approach for fracture prevention and treatment.

[0023] One of the purposes of the present invention is to provide an E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or VEGF-C and uses thereof.

[0024] In a first aspect, the present invention provides a use of an E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or VEGF-C for preparing a composition or preparation for treating fractures and / or promoting fracture healing.

[0025] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN is mammalian E11 / gp38 / podoplanin / PDPN.

[0026] In another preferred embodiment, the VEGF-C is mammalian VEGF-C.

[0027] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0028] In another preferred embodiment, the pharmaceutical composition is in liquid form.

[0029] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liposome preparation.

[0030] In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semisolid composition.

[0031] In another preferred embodiment, the pharmaceutical composition is a liquid composition.

[0032] In another preferred embodiment, the pharmaceutical composition is in the form of an injection or an external-use pharmaceutical formulation.

[0033] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection or a lyophilized preparation.

[0034] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0035] In another preferred embodiment, the preparation includes experimental reagents.

[0036] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitors include: compounds (including small molecule compounds, antibodies, nucleic acids) that directly or indirectly inhibit the activity of E11 / gp38 / podoplanin / PDPN, inhibit the expression of E11 / gp38 / podoplanin / PDPN, and promote the degradation of E11 / gp38 / podoplanin / PDPN.

[0037] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitors include: antibodies, small molecule compounds, microRNA, shRNA, siRNA, antisense oligonucleotides, nucleic acid aptamers, and gene editors.

[0038] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitor includes a pharmaceutically acceptable salt or ester thereof, or an expression vector thereof.

[0039] In another preferred embodiment, the expression vector comprises a viral vector.

[0040] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0041] In another preferred embodiment, the vector is selected from the group consisting of lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof. Preferably, the vector is adeno-associated virus (AAV).

[0042] In another preferred embodiment, the vector comprises AAV2 or AAV9.

[0043] In another preferred embodiment, the composition comprises an anti-podoplanin / PDPN antibody.

[0044] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitors include gene editing reagents and RNA interference (RNAi) reagents.

[0045] In another preferred embodiment, the gene editor includes a DNA gene editor and an RNA gene editor.

[0046] In another preferred embodiment, the gene editor includes optional gRNA and gene editing protein.

[0047] In another preferred example, the gRNA guides the gene editing protein to specifically bind to the RNA of the E11 / gp38 / podoplanin / PDPN gene.

[0048] In another preferred example, the gRNA guides the gene editing protein to specifically bind to the mRNA of the E11 / gp38 / podoplanin / PDPN gene.

[0049] In another preferred embodiment, the composition or preparation is used to treat fractures and / or promote fracture healing.

[0050] In another preferred embodiment, the composition or preparation is used for one or more (or all) applications selected from the following groups:

[0051] (a) Significantly reduce swelling and pain at the fracture end;

[0052] (b) significantly increased BV / TV (bone volume fraction) and Tb.Th (trabecular bone thickness) of callus tissue;

[0053] (c) Significantly increase the maximum stress and yield stress at the fracture healing site.

[0054] In another preferred embodiment, the subject suffering from the bone fracture includes humans and non-human mammals.

[0055] In another preferred embodiment, the non-human mammals include rabbits, rodents (such as mice and rats) and primates (such as monkeys).

[0056] In another preferred embodiment, the mammal does not include a mammal that has undergone ovariectomy.

[0057] In another preferred embodiment, the administration of the E11 / gp38 / podoplanin / PDPN pathway inhibitor includes oral or parenteral administration.

[0058] In another preferred embodiment, the non-oral administration includes (but is not limited to) application.

[0059] The second aspect of the present invention provides a composition or preparation that can be used to treat fractures and / or promote fracture healing, the composition or preparation comprising: (a) a physiologically acceptable carrier; and (b) an effective amount of an active ingredient, wherein the active ingredient is an E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or VEGF-C.

[0060] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitor is selected from the following group: small molecule compounds, antibodies, nucleic acids, or a combination thereof.

[0061] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitor is an antibody.

[0062] In another preferred embodiment, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, or a combination thereof.

[0063] In another preferred embodiment, the antibody is Anti-PDPN.

[0064] In another preferred embodiment, the composition or preparation further contains (c) other active ingredients for treating fractures and / or promoting fracture healing.

[0065] In another preferred embodiment, the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0066] In another preferred embodiment, the composition or preparation further contains a skin penetration enhancer.

[0067] In another preferred embodiment, the physiologically acceptable carrier is selected from the following group: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, nanogels, cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water or a combination thereof.

[0068] In another preferred embodiment, the composition or preparation is in a topical dosage form.

[0069] In another preferred embodiment, the dosage form of the composition or preparation includes ointment, cream, patch, paint, spray, microneedle, etc.

[0070] In another preferred embodiment, the composition or preparation is administered in a manner selected from the group consisting of topical administration, transdermal administration and percutaneous administration.

[0071] The third aspect of the present invention provides a method and / or detection system for in vitro screening of potential substances for treating fractures and / or promoting fracture healing, comprising the steps of:

[0072] (1) adding the substance to be detected or the negative control substance to the detection system; and

[0073] (2) respectively detecting the level of the E11 / gp38 / podoplanin / PDPN pathway in a sample to which the substance to be tested has been added and a sample to which a negative control has been added; if the level of the E11 / gp38 / podoplanin / PDPN pathway in the sample to which the substance to be tested has been added is reduced compared to the sample to which the negative control has been added, the substance is a potential substance for treating fractures and / or promoting fracture healing; or

[0074] The levels of VEGF-C pathway in samples with the addition of the substance to be tested and samples with the addition of a negative control are respectively detected; if the level of VEGF-C pathway in the sample with the addition of the substance to be tested is increased compared with that in the sample with the addition of the negative control, it is a potential substance for treating fractures and / or promoting fracture healing.

[0075] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0076] In another preferred embodiment, except for the different test and control substances added, the experimental conditions of the test group and the control group are the same or substantially the same.

[0077] In another preferred embodiment, the detection system is a cell system.

[0078] A fourth aspect of the present invention provides a method for treating a bone fracture in a subject and / or promoting fracture healing thereof, comprising the steps of:

[0079] A safe and effective amount of the E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or VEGF-C according to the present invention is administered to the subject, thereby treating the subject's fracture and / or promoting fracture healing.

[0080] In another preferred embodiment, the subject includes an animal model.

[0081] In another preferred embodiment, the subject includes a mammal.

[0082] In another preferred embodiment, the subject includes a human.

[0083] In another preferred embodiment, the subject includes non-human mammals.

[0084] In another preferred embodiment, the subject is a non-human mammal.

[0085] In another preferred embodiment, the subject includes a primate.

[0086] In another preferred embodiment, the subject includes a rodent.

[0087] In another preferred embodiment, the subject includes Carnivora.

[0088] In another preferred embodiment, the subject includes animals of the suborder Feliformes.

[0089] In another preferred embodiment, the subject includes Caniformes.

[0090] In another preferred embodiment, the subject includes humans, mice, rats, rabbits, monkeys, dogs, cats, camels, alpacas, or combinations thereof.

[0091] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 shows the effect of Anti-PDPN on soft tissue swelling at the fracture end.

[0093] Figure 2 shows the effect of Anti-PDPN on the thermal pain threshold of the fracture end in fractured mice.

[0094] Figure 3 shows the effect of Anti-PDPN on tibial callus morphometry.

[0095] Figure 4 shows the effect of Anti-PDPN on the biomechanical properties of the tibia.

[0096] Figure 5 shows the effect of Anti-PDPN on lymphoplatelet thrombosis.

[0097] Figure 6 shows the effect of VEGF-C on soft tissue swelling at the fracture end.

[0098] Figure 7 shows the effect of VEGF-C on the thermal pain threshold of the fracture end in fractured mice.

[0099] FIG8 shows the effect of VEGF-C on tibial callus morphometry.

[0100] FIG9 shows the effect of VEGF-C on the biomechanical properties of the tibia. DETAILED DESCRIPTION

[0101] After extensive and in-depth research and extensive screening, the inventors have discovered for the first time that inhibition of the E11 / gp38 / podoplanin / PDPN pathway and / or VEGF-C can help treat fractures and / or promote fracture healing. Experiments have shown that administering anti-PDPN antibodies to subjects can significantly reduce fracture end pain and swelling, significantly increase the BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue, and significantly increase the maximum stress and yield stress at the fracture healing site. Alternatively, administering VEGF-C to subjects can significantly reduce fracture end pain and swelling, significantly increase the BV / TV (bone volume fraction) and Tb.N (trabecular thickness) of callus tissue, and significantly reduce Tb.Sp (trabecular separation), significantly increasing the maximum stress, yield stress, and fracture energy at the fracture healing site. Therefore, the present invention's research unexpectedly and for the first time discovered that inhibitors of the E11 / gp38 / podoplanin / PDPN pathway and / or VEGF-C can treat fractures and / or promote fracture healing, and has broad application prospects. On this basis, the present invention was completed.

[0102] the term

[0103] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0104] For purposes of the description herein and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound.

[0105] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0106] Where a numerical range is provided, it will be understood that, unless the context clearly indicates otherwise, each intermediate integer of that value and each tenth of each intermediate integer of that value (unless the context clearly indicates otherwise), any other specified or intermediate values ​​between the upper and lower limits of the range and in the specified range are included within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also encompassed within the present invention, subject to any explicitly excluded limitations within the specified range. When a specified range includes one or two of these limits, ranges excluding (i) either one or (ii) both of those included limits are also included within the present invention. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0107] Generally, the nomenclature used herein and the techniques and procedures described herein include those well known and commonly used by those of ordinary skill in the art, such as the common techniques and methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

[0108] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0109] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0110] As used herein, the term "administering" refers to physically introducing a product of the disclosure into a subject, such as by injection or infusion, using any of a variety of methods and delivery systems, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration.

[0111] As used herein, the term "antibody (Ab)" includes, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with the antigen.

[0112] E11 / gp38 / podoplanin / PDPN pathway inhibitors

[0113] In the present invention, the term "active ingredient" or "active ingredient for treating bone fractures and / or promoting fracture healing" refers to an antagonist or inhibitor of the E11 / gp38 / podoplanin / PDPN pathway.

[0114] In the present invention, representative inhibitors of the E11 / gp38 / podoplanin / PDPN pathway include (but are not limited to): small molecule compounds, antibodies, nucleic acids, or combinations thereof. Preferred examples include compounds and antibodies that specifically target proteins in the E11 / gp38 / podoplanin / PDPN pathway.

[0115] As used herein, the terms "E11 / gp38 / podoplanin / PDPN inhibitor" and "E11 / gp38 / podoplanin / PDPN pathway inhibitor" are used interchangeably and refer to molecules that can interfere with the activity of E11 / gp38 / podoplanin / PDPN. The E11 / gp38 / podoplanin / PDPN inhibitor can be an anti-E11 / gp38 / podoplanin / PDPN antibody that specifically binds to the E11 / gp38 / podoplanin / PDPN protein and inhibits its activity. In addition, the E11 / gp38 / podoplanin / PDPN inhibitor can be an inhibitory nucleic acid. The inhibitory nucleic acid can be an aptamer that specifically binds to the E11 / gp38 / podoplanin / PDPN protein and inhibits its activity. Other inhibitory nucleic acids can bind to the E11 / gp38 / podoplanin / PDPN transcript and inhibit its translation or degrade it. Typically, such inhibitory nucleic acids can be antisense nucleic acids, morpholino oligonucleotides, inhibitory RNA molecules such as siRNA or microRNA, or ribozymes. Furthermore, E11 / gp38 / podoplanin / PDPN inhibitors can be small molecules that bind to E11 / gp38 / podoplanin / PDPN and inhibit its activity. Such small molecule inhibitors of E11 / gp38 / podoplanin / PDPN can be obtained by well-known screening procedures or molecular modeling methods, the goal of which is to identify compounds that bind to the active site of the E11 / gp38 / podoplanin / PDPN domain.

[0116] In some embodiments, the E11 / gp38 / podoplanin / PDPN inhibitor comprises an antibody, including, but not limited to, a humanized antibody, a monoclonal antibody, a polyclonal antibody, a monovalent antibody, a bivalent antibody, a multivalent antibody, an immunoconjugate, and an antibody-drug conjugate.

[0117] As used herein, "antibody" refers to a molecule comprising one or more polypeptide chains that specifically binds to or is immunoreactive with a specific antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (or heterologous conjugate) antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., one-armed antibodies), multivalent antibodies, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0118] An "anti-E11 / GP38 / PODOPLANIN / PDPN antibody" or "antibody that binds to E11 / GP38 / PODOPLANIN / PDPN" refers to an antibody that binds to E11 / GP38 / PODOPLANIN / PDPN with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting E11 / GP38 / PODOPLANIN / PDPN. In some embodiments, the extent of binding of an anti-E11 / GP38 / PODOPLANIN / PDPN antibody to an unrelated, non-E11 / GP38 / PODOPLANIN / PDPN antigen is less than about 10% of the binding of the antibody to E11 / GP38 / PODOPLANIN / PDPN, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that binds E11 / GP38 / PODOPLANIN / PDPN has a dissociation constant (KD) of <1 pM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10-8 M or less, e.g., 10-8 M to 10-13 M, e.g., 10-9 M to 10-13 M).

[0119] "Full length antibody," "intact antibody," or "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

[0120] An "antibody fragment" is a portion of a full-length antibody that is capable of binding to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; monovalent or single-arm antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0121] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these antibodies are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and α, respectively.

[0122] "Variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that is involved in binding the antibody to the antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (CDR) (see, e.g., Kindt et al., Ruby Immunology, 6th edition, WH Freeman and Co., page 91). A single VH or VL domain may be sufficient to confer specificity for antigen binding. In addition, the VH or VL domain of an antibody that binds to an antigen can be used to screen a library of complementary VL or VH domains, respectively, to isolate antibodies that bind to a specific antigen.

[0123] As used herein, "hypervariable region" or "CDR" refers to each region of an antibody variable domain whose sequence is highly variable and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural antibody comprises four chains with six CDRs; three in the heavy chain variable domain VH (CDR-H1, CDR-H2, CDR-H3) and three in the light chain variable domain VL (CDR-L1, CDR-L2, CDR-L3). CDRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs). Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Rabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0124] As used herein, "complementarity determining region" or "CDR" refers to the region within the CDRs of the variable domain with the highest sequence variability and / or involved in antigen recognition. Typically, natural antibodies comprise four chains with six CDRs; three in the heavy chain variable domain VH (H1, H2, H3) and three in the light chain variable domain VL (L1, L2, L3). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 26-35 or 31-35 of H1, 50-65 or 50-65 of H2, and 93-102 or 95-102 of H3. (Rabat et al., supra). Alternative methods to Rabat et al. (supra) have been proposed to collect and align sequences of different members of the immunoglobulin superfamily, such as Chothia et al. (Chothia - J Mol Biol. 1987 Aug 20; 196(4): 901-17 and Nature. 1989 Dec 21-28; 342(6252): 877-83.) and Lefranc et al. (IMGT - Nucleic Acids Res. 1999 Jan 1; 27(1): 209-12.), who proposed a unified numbering scheme for immunoglobulin variable domain germline sequences. The present invention encompasses all such alternative definitions, and the sequences provided in this specification are not intended to exclude alternatively defined CDR sequences that may comprise only a portion of the CDR sequences provided in the sequence listing. Specifically, the CDR sequences according to Chothia et al. occur at amino acid residues 26–31 of H1, 52–65 of H2, and 95–102 of H3;

[0125] Specifically, the CDR sequences according to LeFranc et al. occur at amino acid residues 27-38 of H1, 56-65 of H2, and 105-117 of H3.

[0126] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (CDR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0127] "Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains linked by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be divided into one of two types, referred to as kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0128] As used herein, " monoclonal antibody " refers to the antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is identical and / or in conjunction with the same epi-position, except possible variant antibodies (for example, variant antibodies contain naturally occurring or mutations occurring during the monoclonal antibody production process, and are generally present in small amounts). In contrast to the polyclonal antibody preparations that generally include different antibodies for different determinants (epi-topes), every kind of monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the term " monoclonal " represents the feature of the antibody obtained from a substantially homogeneous antibody population, and should not be construed as needing to produce antibodies by any ad hoc method. For example, monoclonal antibody to be used can be prepared by various techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method and the method for utilizing a transgenic animal containing all or part of a human immunoglobulin locus, and these methods and other exemplary methods for the preparation of monoclonal antibodies are described herein.

[0129] A "chimeric antibody" is an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0130] "Humanized antibody" refers to a chimeric antibody comprising an amino acid sequence from a non-human CDR and an amino acid sequence from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one and generally two variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0131] A "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0132] A "human consensus framework" is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup such as Rabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI such as Rabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III such as Rabat et al. (supra).

[0133] As used herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0134] "Fc region" refers to a dimer complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is a sequence that can be obtained by digesting a complete antibody with papain. The Fc region can comprise a native or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence is generally defined as extending from about the amino acid residue at position Cys226 or about the amino acid residue at position Pro230 to the carboxyl terminus of the Fc sequence at Lys447. However, due to enzymatic cleavage that may occur in a cell culture system for recombinant production (e.g., production in CHO cells), the C-terminal lysine (Lys447) of the Fc sequence may be present or absent in the Fc region of the recombinant antibody. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain.

[0135] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcR is a natural human FcR. In some embodiments, FcR is an FcR that binds to IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences, differing mainly in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.

[0136] As used herein, FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of parental IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., Eur. J. Immunol., 24:2429-2434 (1994)) and regulating the homeostasis of immunoglobulins. FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol, 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994) and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995).

[0137] As used herein, a "multivalent antibody" is an antibody that contains three or more antigen binding sites. A multivalent antibody is preferably engineered to have three or more antigen binding sites and is typically not a native sequence IgM or IgA antibody.

[0138] A "multispecific antibody" is an antibody that has at least two different binding sites, each with a different binding specificity. A multispecific antibody can be a full-length antibody or an antibody fragment, and the different binding sites can each bind to a different antigen, or the different binding sites can bind to two different epitopes of the same antigen.

[0139] "Fv fragment" refers to an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain and a light chain variable domain that are tightly bound, which can be naturally covalent, such as in scFv. It is in this configuration that the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six CDRs or their subgroups together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is usually lower than that of the entire binding site.

[0140] A "Fab fragment" refers to an antibody fragment containing the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. A "F(ab')2 fragment" comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0141] As used herein, "antigen binding arm" refers to the component of an antibody that has the ability to specifically bind to a target molecule of interest. Typically, an antigen binding arm is a complex of immunoglobulin polypeptide sequences, e.g., CDRs and / or variable domain sequences of immunoglobulin light and heavy chains.

[0142] "Single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired antigen-binding structure.

[0143] "Diabodies" refer to small antibody fragments with two antigen-binding sites, which contain a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0144] "Linear antibodies" refer to the antibodies described in Zapata et al. Protein Eng., 8(10): 1057-1062 (1995). Briefly, these antibodies contain tandem repeats of heavy chain fragments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0145] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel.

[0146] "Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). "Binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0147] "Specifically binds" or "specific binding" refers to the binding of an antibody to an antigen with an affinity value of no more than about 1 x 10-7 M.

[0148] An "affinity matured" antibody is one with one or more alterations in one or more CDRs compared to a parent antibody that does not possess such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

[0149] A "functional antigen-binding site" of an antibody is one that is capable of binding to a target antigen. The antigen-binding affinity of an antigen-binding site is not necessarily as strong as that of the parent antibody from which the antigen-binding site is derived, but the ability to bind antigen must be measurable using any of a variety of methods known for assessing antibody-antigen binding.

[0150] An "isolated antibody" refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reversed-phase HPLC).

[0151] Composition or preparation

[0152] The present invention provides a composition or preparation that can be used to treat fractures and / or promote fracture healing, comprising: (a) an acceptable carrier (including a pharmaceutically acceptable carrier, a physiologically acceptable carrier, and a cosmetically acceptable carrier); and (b) an effective amount of an active ingredient, wherein the active ingredient is an E11 / gp38 / podoplanin / PDPN pathway inhibitor.

[0153] In the present invention, the effective amount of the active ingredient may vary depending on the mode of use and the specific conditions of the desired subject, etc. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials).

[0154] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0155] As used herein, the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0156] The term "physiologically acceptable carrier" refers to a carrier for the active ingredient, including: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water or a combination thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0157] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be compatible with the mode of administration. The pharmaceutical compositions of the present invention are available in the form of injections, oral preparations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they can be prepared using conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0158] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0159] In the present invention, the dosage forms of the composition or preparation include (but are not limited to): solid preparations, liquid preparations, gel preparations, semi-solid preparations, and some preferred dosage forms include ointments, creams, patches, paints, sprays, microneedles, solutions, etc.

[0160] In another preferred embodiment, the composition or preparation is administered in a manner selected from the group consisting of topical administration, transdermal administration and percutaneous administration.

[0161] Representative routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, inhalation and parenteral (intravenous, intramuscular or subcutaneous).

[0162] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0163] Combination medicines for inhalation may include aerosols, sprays, and the like.

[0164] The composition of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0165] When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds can be administered simultaneously, separately, or sequentially with the compound of the present invention.

[0166] The main advantages of the present invention include:

[0167] (a) It can significantly reduce the swelling and pain at the fracture ends;

[0168] (b) It can significantly increase the BV / TV (bone volume fraction) and Tb.Th (trabecular bone thickness) of callus tissue;

[0169] (c) The maximum stress, yield stress, and / or fracture energy at the fracture healing site can be significantly increased.

[0170] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0171] Materials and Methods

[0172] 1. Experimental Animals

[0173] A total of 80 SPF-grade male C57BL / 6 mice, aged 6-8 weeks, were purchased from Shanghai JCSJ Laboratory Animal Co., Ltd. (Shanghai) Animal Certification Number: SCXK(Shanghai)2018-0004 and housed at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The temperature and humidity in the housing room were controlled at 23°C and 60%, respectively, with adequate sunlight, mouse chow, and drinking water. This study was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine, with the ethics approval number PZSHUTCM211101023.

[0174] 2. Experimental drugs and preparation methods

[0175] Anti-mouse Podoplanin (BioXcell, BE0236, USA), Polyclonal Syrian hamster IgG (BioXcell, BP0087, USA), isoflurane (Voraid, China).

[0176] Preparation of 1 mg / ml IgG isotype antibody: Dissolve 0.2 ml of 1.7 mg IgG protein in 1500 μl of sterile PBS solution.

[0177] Preparation of 1mg / ml Anti-PDPN antibody: Dissolve 0.7ml of 1.5mg IgG protein in 800ul sterile PBS solution.

[0178] Cys156Ser (R&D Systems, 752-VC-025 / CF, USA), InVivoPure dilution buffer (Bio X Cell, IP0070, USA), isoflurane (Voraid, China).

[0179] Preparation of VEGF-C solution: 25ug Cys156Ser was dissolved in 150ul InVivoPure dilution buffer.

[0180] 3. Experimental equipment

[0181] vivaCT40 mouse Micro-CT scanner (Scanco Medical AG, Switzerland); multi-channel small animal gas anesthesia machine (Matrx VMR, USA), microinjectors (HAMILTON, China), 1 ml sterile syringes (0.45 × 16 mm, Shandong Weigao), ElectroForce 3200 biomechanics machine (TA-Bose, USA), pain threshold machine (Ugo Basile, Italy), and a homemade soft tissue swelling meter.

[0182] 4. Modeling

[0183] Method for establishing a right tibial fracture with combined intramedullary fixation: After isoflurane anesthesia, mice were placed in the supine position. A longitudinal incision approximately 1.5 cm below the midpoint of the right knee was made to expose the lateral margin of the mid-tibia. The surrounding tibial muscles were bluntly dissected to avoid damaging the lymphatic vessels and lymph nodes draining the hind limb. A 1 ml sterile syringe needle was inserted from the midpoint of the tibial plateau and along the long axis of the tibia into the medullary cavity approximately 1.5 cm. The needle was withdrawn 0.2 cm proximal to the tibial plateau. The mid-tibia was gently severed with a blade. After the fracture ends were aligned, the needle was lowered into the medullary cavity for intramedullary fixation. When the intramedullary needle was in close contact with the inner wall of the bone medullary cavity, it was gently rotated clockwise or counterclockwise. Good alignment and successful modeling were confirmed by visible rotation of the ankle joint in the same direction. The needle cap exposed on the tibial plateau was cut off, the skin was sutured, and antibacterial ointment was applied to prevent infection.

[0184] 5 detection indicators

[0185] 5.1 Detection of soft tissue swelling at the fracture site using a modified limb swelling meter Before modeling and on days 1, 2, 3, 5, and 7 after successful modeling, the right hind limb of the mouse below the knee joint was completely immersed in a new practical swelling meter. The average value of the three measurements was recorded as the experimental result. The soft tissue swelling at the fracture site was calculated as the swelling volume after modeling / the swelling volume before modeling*100%.

[0186] 5.2 Plantar Thermal Pain Threshold Assay: On the second day after model establishment, mice, previously deprived of food and water, were placed in a closed chamber within the measuring instrument and allowed to acclimate for 2 hours. After rest, 30% radiant heat was applied to induce a paw withdrawal reaction. When the mouse experienced pain and raised its hind paw, the instrument automatically recorded the corresponding reaction time of the paw withdrawal. Each mouse was tested five times. After discarding the highest and lowest values ​​from the five measurements, the average of the remaining three values ​​was calculated as the average reaction time for each mouse.

[0187] 5.3 Micro-CT Scanning: Tibial tissue specimens from mice treated with 75% ethanol were placed in a micro-CT chamber and X-rayed to observe tibial fracture healing and callus formation in each group. Subsequently, CT imaging of the tibia specimens (between the tibial plateau and the ankle joint) was performed using the following parameters: voltage 70 kV, current 142 μA, 9 μm resolution, and a 0.5 mm aluminum filter. Three-dimensional reconstruction of bone tissue morphology and callus trabecular parameters were performed using the accompanying software.

[0188] 5.4 Three-Point Bending Test After sampling, carefully remove all attached muscle from the tibial specimens, wrap them in saline-soaked gauze, and store them at 4°C. Once all specimens are collected, place them flat in the mechanical analysis machine, maintaining a uniform angle between the calluses and the center of the tester. Lock the specimens at both ends, open the computer setup program, and begin the analysis.

[0189] 6. Statistical Analysis

[0190] Statistical data were presented as mean ± standard deviation (SD) and analyzed using SPSS 20.0. If two independent data sets followed a normal distribution and had homogeneous variances, a t-test was used; if the variances were unequal, a t' test was used. If two independent data sets did not follow a normal distribution, a nonparametric Mann-Whitney U test was used. Repeated-measures analysis of variance and simple-effects tests were used for data from two groups measured at multiple time points. The test level was α = 0.05, and P < 0.05 was considered statistically significant.

[0191] Example 1: Effect of Anti-PDPN on Fractures in Mice

[0192] Mice were randomly divided into IgG group and Anti-PDPN group after labeling using random number table method, with 20 mice in each group. Intervention was performed 2 days before modeling. IgG group: 25ul / time of IgG isotype antibody was injected locally around the popliteal lymph nodes under B-ultrasound guidance 2 days before modeling. The injection was given once every 2 days for 4 consecutive times. Anti-PDPN group: 25ul / time of anti-Podolanin antibody was injected locally around the popliteal lymph nodes under B-ultrasound guidance 2 days before modeling. The injection was given once every 2 days for 4 consecutive times. The index test results are as follows:

[0193] As shown in Figure 1, on day 2 after fracture, mice in the IgG group showed significant soft tissue swelling in the tibia and ankle joint on the fracture side, while mice in the Anti-PDPN group showed only slight soft tissue swelling in the tibia. Using a modified limb swelling meter, soft tissue swelling at the fracture site of the mice was measured. On days 1, 2, 3, 5, and 7 after modeling, soft tissue swelling in the fracture-side hindlimb of the Anti-PDPN group was significantly lower than that in the vehicle group (P < 0.05).

[0194] As shown in Figure 2 , the thermal pain threshold of the plantar surface of the two groups of mice was tested on the second day after modeling. The thermal pain threshold of the Anti-PDPN group was significantly lower than that of the solvent group (P<0.05).

[0195] As shown in Figure 3, micro-CT reconstruction and analysis of tibial callus tissue 14 days after fracture in mice revealed that mice in the Anti-PDPN group had larger callus formation at the fracture site with a blurred fracture line, while mice in the IgG group had smaller callus formation at the fracture site with a clearer fracture line. Bone mass parameters of tibial callus tissue in each group 14 days after fracture showed that BV / TV and Tb.Th values ​​were significantly increased in the Anti-PDPN group compared with the IgG group (P < 0.01), but Tb.N and Tb.Sp values ​​were not significantly different between the two groups (P > 0.05). This suggests that the Anti-PDPN group exhibited superior tibial callus morphometry compared with the vehicle group.

[0196] As shown in Figure 4 , biomechanical testing was performed on the 35th day after modeling. The maximum stress and yield stress of the callus of mice in the Anti-PDPN group were significantly higher than those in the IgG group (P<0.05), but there was no significant difference in fracture energy (P>0.05).

[0197] It is generally shown that Anti-PDPN can reduce swelling and pain after fracture and promote fracture healing.

[0198] Example 2: Effect of Anti-PDPN on Lymph Platelet Thrombosis

[0199] To verify whether Anti-PDPN can prevent lymphoplatelet thrombosis (LPT), thereby improving lymphatic drainage and fracture healing, Anti-PDPN was locally injected into the popliteal lymph nodes (PLN) of mice 2 days before modeling and continued until 7 days after fracture, when treatment was stopped. On the 7th day after fracture, lymphatic morphology was measured using NIR-ICG, and PLNs of hindlimb fractures were harvested and stained using immunofluorescence to assess the number and coverage of LPTs.

[0200] The results are shown in Figure 5. Compared with the IgG control group, the Anti-PDPN group significantly reduced the expression of CD41 + These results indicate that Anti-PDPN can inhibit the formation of lymphatic platelet thrombus and dredge lymphatic vessels, thereby promoting fracture healing.

[0201] Example 3: Effect of VEGF-C on bone fractures in mice

[0202] Mice were randomly divided into a solvent group (VEH group) and a VEGF-C group using a random number table, with 20 mice in each group. After successful model establishment, ① the solvent group: immediately after model establishment, the fracture end was injected intramuscularly with 6ul of InVivoPure dilution buffer / week / time for 2 or 5 weeks. ② the VEGF-C group: immediately after model establishment, the fracture end was injected intramuscularly with 6ul of VEGF-C solution / week / time for 2 or 5 weeks. The index test results are as follows:

[0203] As shown in Figure 6, on day 2 after fracture, the solvent group showed significant soft tissue swelling in the tibia and ankle joint on the fracture side, as well as small ulcers on the skin surface; whereas the VEGF-C group showed only slight soft tissue swelling in the tibia. Using a modified limb swelling meter to measure soft tissue swelling at the fracture site, the VEGF-C group showed significantly lower soft tissue swelling in the fracture-side hindlimb than the solvent group on days 1 and 3 after modeling (P < 0.05).

[0204] As shown in FIG7 , the thermal pain threshold of the plantar surface of the two groups of mice was tested on the second day after modeling. The thermal pain threshold of the VEGF-C group was significantly lower than that of the solvent group (P<0.01).

[0205] As shown in Figure 8, Micro-CT reconstruction and analysis of tibial callus tissue 14 days after fracture in mice revealed that mice in the VEGF-C group had larger callus formation at the fracture site, with a blurred fracture line, while mice in the VEH group had less callus formation and a clearer fracture line. Analysis of bone mass parameters in the tibial callus tissue of each group 14 days after fracture revealed a significant increase in BV / TV and Tb.N, while a significant decrease in Tb.Sp, in the VEGF-C group compared with the vehicle group (both P < 0.05). Tb.Th was not significantly different from the VEH group (P > 0.05). This suggests that the VEGF-C group exhibited superior tibial callus morphometry compared with the vehicle group.

[0206] As shown in Figure 9 , biomechanical tests were performed on the 35th day after modeling, and the maximum stress, yield stress, and fracture energy of the callus of mice in the VEGF-C group were significantly higher than those in the VEH group (P<0.05).

[0207] The above experimental results comprehensively indicate that VEGF-C can reduce swelling and pain after fracture and promote fracture healing, including significantly reducing swelling and pain at the fracture ends of mice, significantly increasing the BV / TV (bone volume fraction) and Tb.N (trabecular thickness) of callus tissue and significantly reducing Tb.Sp (trabecular separation), and significantly increasing the maximum stress, yield stress and fracture energy at the fracture healing site; thus, it can be used to prepare drugs for the treatment of fractures.

[0208] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of an active ingredient, characterized in that: A composition or preparation for treating bone fractures and / or promoting bone fracture healing; the active ingredient is selected from the following group: (1) E11 / gp38 / podoplanin / PDPN pathway inhibitors; and / or (2) Vascular endothelial growth factor C (VEGF-C).

2. The use according to claim 1, characterized in that The E11 / gp38 / podoplanin / PDPN pathway inhibitors include antibodies, small molecule compounds, microRNA, shRNA, siRNA, antisense oligonucleotides, nucleic acid aptamers, and gene editors.

3. The use according to claim 1, characterized in that The E11 / gp38 / podoplanin / PDPN pathway inhibitor is an anti-podoplanin / PDPN antibody.

4. A composition or preparation for treating bone fractures and / or promoting bone fracture healing, characterized in that: The composition or preparation comprises: (a) a physiologically acceptable carrier; and (b) an effective amount of an active ingredient, wherein the active ingredient is an E11 / gp38 / podoplanin / PDPN pathway inhibitor and / or VEGF-C.

5. The composition or preparation according to claim 4, characterized in that The physiologically acceptable carrier is selected from the following group: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, nanogels, cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water or a combination thereof.

6. The composition or preparation according to claim 4, characterized in that The dosage form of the composition or preparation is a topical dosage form.

7. The composition or preparation according to claim 4, characterized in that The dosage forms of the composition or preparation include ointment, cream, patch, paint, spray, microneedle and the like.

8. The composition or preparation according to claim 4, characterized in that The composition or preparation is administered in a manner selected from the group consisting of topical administration, transdermal administration and percutaneous administration.

9. The composition or preparation according to claim 4, characterized in that The composition or preparation is used for treating bone fractures and / or promoting bone fracture healing.

10. The composition or preparation according to claim 4, characterized in that The composition or preparation is used for one or more applications selected from the following group: (a) Significantly reduce swelling and pain at the fracture ends; (b) Significantly increased BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue; (c) Significantly increase the maximum stress and yield stress at the fracture healing site.

11. A method and / or detection system for in vitro screening of potential substances for treating fractures and / or promoting fracture healing, characterized in that: Includes steps: (1) adding the substance to be detected or the negative control substance to the detection system respectively; and (2) respectively detecting the level of E11 / gp38 / podoplanin / PDPN pathway in the sample added with the substance to be tested and the sample added with the negative control; if the level of E11 / gp38 / podoplanin / PDPN pathway in the sample added with the substance to be tested is reduced compared with the sample added with the negative control, it is a potential substance for treating bone fractures and / or promoting bone fracture healing; or The levels of VEGF-C pathway in the sample with the substance to be tested and the sample with the negative control are detected respectively; if the level of VEGF-C pathway in the sample with the substance to be tested increases compared with that in the sample with the negative control, it is a potential substance for treating fractures and / or promoting fracture healing.

12. The method according to claim 11, characterized in that: The method is non-diagnostic and non-therapeutic.

13. The method according to claim 11, characterized in that: Except for the differences in the added test and control substances, the experimental conditions of the test group and the control group are the same or substantially the same.

14. The method according to claim 11, characterized in that: The detection system is a cell system.

15. A method for treating a bone fracture in a subject in need thereof and / or promoting fracture healing, comprising the steps of: A safe and effective amount of an inhibitor of the E11 / gp38 / podoplanin / PDPN pathway and / or VEGF-C is administered to the subject, thereby treating the subject's fracture and / or promoting fracture healing.

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