E11 / gp38 / podoplanin / PDPN pathway inhibitor and application thereof

Abstract

The invention relates to an application of an E11 / gp38 / podoplanin / PDPN pathway inhibitor in preparation of a medicine for treating fracture and / or promoting fracture healing. The invention also discloses a pharmaceutical preparation for treating fracture and / or promoting fracture healing. Experiments show that the inhibitor disclosed by the invention can be used for remarkably relieving mouse fracture broken end swelling and pain, remarkably improving BV / TV (bone volume fraction) and Tb.Th (bone trabecula thickness) of callus tissues, and remarkably improving the maximum stress and yield stress of fracture healing parts. The inhibitor can be used for preparing medicines for treating fracture.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of an E11 / gp38 / podoplanin / PDPN pathway inhibitor in the preparation of a medicine for treating fractures. Background Art

[0002] Traumatic fractures are common diseases in the field of orthopedics. Even if clinical treatment follows the three principles of "good fixation, blood circulation and anti-infection", the incidence of delayed fracture healing and nonunion is still as high as 5-10%, and fractures of the femur and tibia and fibula are the most common, which not only brings disability and pain to patients, but also brings a heavy burden to the entire family and society. At present, the main method of promoting fracture repair in Western medicine is to enhance bone conduction and bone induction functions, such as bone transplant substitutes and bone growth factors, but the cost is expensive and the efficacy is uncertain.

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

[0004] 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 drugs for preventing and treating fractures and / or promoting fracture healing.

[0005] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, characterized in that Anti-PDPN is used to reduce soft tissue swelling at the fracture ends of mice.

[0006] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, characterized in that Anti-PDPN is used to reduce the thermal pain threshold of mice.

[0007] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, characterized in that Anti-PDPN is used to increase the BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue.

[0008] Furthermore, Anti-PDPN is used in the preparation of drugs for preventing and treating fractures and / or promoting fracture healing, wherein Anti-PDPN is used to increase the maximum stress and yield stress at the fracture healing site.

[0009] Furthermore, Anti-PDPN is used in the preparation of a drug for preventing and treating fractures and / or promoting fracture healing, which is characterized by comprising a therapeutically effective amount of Anti-PDPN or Anti-PDPN and a pharmaceutically acceptable excipient.

[0010] Anti-PDPN is used to prepare a pharmaceutical preparation for preventing and treating fractures and / or promoting fracture healing, characterized in that 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.

[0011] 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.

[0012] 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 the Anti-podoplanin neutralizing antibody can significantly reduce the swelling and pain of 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, the Anti-podoplanin neutralizing antibody provides a new way to prevent and treat fractures.

[0013] One of the purposes of the present invention is to provide an E11 / gp38 / podoplanin / PDPN pathway inhibitor and its application.

[0014] In a first aspect, the present invention provides a use of an E11 / gp38 / podoplanin / PDPN pathway inhibitor for preparing a composition or preparation for treating bone fractures and / or promoting bone fracture healing.

[0015] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN is mammalian E11 / gp38 / podoplanin / PDPN.

[0016] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0017] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid dosage form.

[0018] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liposome preparation.

[0019] In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semisolid composition.

[0020] In another preferred embodiment, the pharmaceutical composition is a liquid composition.

[0021] In another preferred embodiment, the pharmaceutical composition is in the form of an injection or an external-use pharmaceutical dosage form.

[0022] In another preferred embodiment, the dosage form of the pharmaceutical composition includes an injection or a lyophilized preparation.

[0023] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0024] In another preferred embodiment, the preparation includes experimental reagents.

[0025] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitor includes: 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.

[0026] 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.

[0027] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitor includes a pharmaceutically acceptable salt or ester thereof, or an expression vector thereof.

[0028] In another preferred embodiment, the expression vector comprises a viral vector.

[0029] In another preferred embodiment, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

[0030] In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof. Preferably, the vector is adeno-associated virus (AAV).

[0031] In another preferred embodiment, the vector comprises AAV2 or AAV9.

[0032] In another preferred embodiment, the composition comprises an anti-podoplanin / PDPN antibody.

[0033] In another preferred embodiment, the E11 / gp38 / podoplanin / PDPN pathway inhibitors include gene editing reagents and RNA interference (RNAi) reagents.

[0034] In another preferred embodiment, the gene editor includes a DNA gene editor and an RNA gene editor.

[0035] In another preferred embodiment, the gene editor includes optional gRNA and gene editing protein.

[0036] In another preferred example, the gRNA guides the gene editing protein to specifically bind to the RNA of the E11 / gp38 / podoplanin / PDPN gene.

[0037] In another preferred example, the gRNA guides the gene editing protein to specifically bind to the mRNA of the E11 / gp38 / podoplanin / PDPN gene.

[0038] In another preferred embodiment, the composition or preparation is used to treat fractures and / or promote fracture healing.

[0039] In another preferred embodiment, the composition or preparation is used for one or more (or all) applications selected from the following groups:

[0040] (a) Significantly reduce swelling and pain at the fracture ends;

[0041] (b) Significantly increased BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue;

[0042] (c) Significantly increase the maximum stress and yield stress at the fracture healing site.

[0043] In another preferred embodiment, the subject suffering from the bone fracture includes humans and non-human mammals.

[0044] In another preferred embodiment, the non-human mammals include rabbits, rodents (such as mice, rats) and primates (such as monkeys).

[0045] In another preferred embodiment, the mammals do not include mammals that have undergone ovariectomy.

[0046] In another preferred embodiment, the administration of the E11 / gp38 / podoplanin / PDPN pathway inhibitor includes oral or parenteral administration.

[0047] In another preferred embodiment, the non-oral administration includes (but is not limited to) application.

[0048] 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.

[0049] 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.

[0050] In another preferred embodiment, the composition or preparation further contains (c) other active ingredients for treating fractures and / or promoting fracture healing.

[0051] 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 when necessary.

[0052] In another preferred embodiment, the composition or preparation further contains a skin penetration enhancer.

[0053] 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, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water or a combination thereof.

[0054] In another preferred embodiment, the composition or preparation is in a topical dosage form.

[0055] In another preferred embodiment, the dosage form of the composition or preparation includes ointment, cream, patch, paint, spray, microneedle, etc.

[0056] 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.

[0057] 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:

[0058] (1) adding the substance to be detected or the negative control substance to the detection system respectively; and

[0059] (2) respectively detecting the levels of the E11 / gp38 / podoplanin / PDPN pathway in samples to which the substance to be tested has been added and samples to which the negative control has been added; if the levels of the E11 / gp38 / podoplanin / PDPN pathway in samples to which the substance to be tested has been added are reduced compared to samples to which the negative control has been added, the substance is a potential substance for treating fractures and / or promoting fracture healing.

[0060] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0061] 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.

[0062] In another preferred embodiment, the detection system is a cell system.

[0063] A fourth aspect of the present invention provides a method for treating a bone fracture in a subject in need thereof and / or promoting fracture healing thereof, comprising the steps of:

[0064] A safe and effective amount of the E11 / gp38 / podoplanin / PDPN pathway inhibitor of the present invention is administered to the subject, thereby treating the fracture of the subject and / or promoting the fracture healing thereof.

[0065] In another preferred embodiment, the subject includes an animal model.

[0066] In another preferred embodiment, the subject includes a mammal.

[0067] In another preferred embodiment, the subject includes a human.

[0068] In another preferred embodiment, the subject includes non-human mammals.

[0069] In another preferred embodiment, the subject is a non-human mammal.

[0070] In another preferred embodiment, the subject includes primates.

[0071] In another preferred embodiment, the subject includes a rodent.

[0072] In another preferred embodiment, the subject includes Carnivora.

[0073] In another preferred embodiment, the object includes animals of the suborder Felidae.

[0074] In another preferred embodiment, the object includes Caniformes.

[0075] In another preferred embodiment, the subject includes humans, mice, rats, rabbits, monkeys, dogs, cats, camels, alpacas, or combinations thereof.

[0076] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The effect of Anti-PDPN on soft tissue swelling at fracture ends.

[0078] Figure 2 This is the effect of Anti-PDPN on the thermal pain threshold of the fracture end in fractured mice.

[0079] Figure 3 The effect of Anti-PDPN on the morphometry of tibial callus.

[0080] Figure 4 The effect of Anti-PDPN on the biomechanical properties of the tibia. DETAILED DESCRIPTION

[0081] After extensive and in-depth research and a large number of screenings, the inventors discovered for the first time that inhibition of the E11 / gp38 / podoplanin / PDPN pathway can help treat fractures and / or promote fracture healing. Experiments have shown that by administering anti-PDPN antibodies to subjects, it is possible to: significantly reduce swelling and pain at fracture ends, 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. Therefore, the research of the present invention unexpectedly discovered for the first time that E11 / gp38 / podoplanin / PDPN pathway inhibitors have the effect of treating fractures and / or promoting fracture healing, and have broad application prospects. On this basis, the present invention was completed.

[0082] the term

[0083] 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.

[0084] For purposes of the description herein and the claims that follow, 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.

[0085] 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.).

[0086] Where a numerical range is provided, it is to be understood that each intermediate integer of the value and each tenth of each intermediate integer of the value (unless the context clearly indicates otherwise), between the upper and lower limits of the range and any other specified or intermediate values ​​in the specified range are included in the present invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed in the present invention, subject to any explicitly excluded limitations within the specified ranges. When the specified range includes one or two of these limits, the range excluding (i) any one or (ii) two of those included limits is also included in the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0087] Generally, the terms used herein and the techniques and procedures described herein include those well known and commonly used by ordinary technicians in the art, such as the common techniques and methods described in the following documents: Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter referred to as "Sambrook"); Current Protocols in Molecular Biology, FM Ausubel et al., ed., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented to 2011) (hereinafter referred to as "Ausubel"); Antibody Engineering, Volumes 1 and 2, R. Kontermann and S. Dubel, ed., Springer-Verlag, Berlin and Heidelberg (2010); Monoclonal Antibodies: Methods and Protocols, V. Ossipow and N. Fischer, ed., 2nd Edition, Humana Press (2014); Therapeutic Antibodies: From Bench to Clinic, edited by Z. An, J. Wiley & Sons, Hoboken, NJ (2009); and Phage Display, edited by Tim Clackson and Henry B. Lowman, Oxford University Press, United Kingdom (2004).

[0088] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0089] 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.

[0090] 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.

[0091] As used herein, the term "antibody (Ab)" includes, but is not limited to, an immunoglobulin that specifically binds to an antigen and comprises 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 complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens.

[0092] E11 / gp38 / podoplanin / PDPN pathway inhibitors

[0093] In the present invention, the term "active ingredient" or "active ingredient for treating bone fractures and / or promoting bone fracture healing" refers to an antagonist or inhibitor of the E11 / gp38 / podoplanin / PDPN pathway.

[0094] 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.

[0095] 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. In addition, 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 purpose of which is to identify compounds that bind to the active site of the E11 / gp38 / podoplanin / PDPN domain.

[0096] In some embodiments, the E11 / gp38 / podoplanin / PDPN inhibitor comprises an antibody, including but not limited to humanized antibodies, monoclonal antibodies, polyclonal antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, immunoconjugates, and antibody-drug conjugates.

[0097] 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 heteroconjugated) 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).

[0098] "Anti-E11 / GP38 / PODOPLANIN / PDPN Antibody" or "Binding

[0099] An "antibody against 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 anti-E11 / GP38 / PODOPLANIN / PDPN antibody is bound to an unrelated non-

[0100] The antibody binds less to the E11 / GP38 / PODOPLANIN / PDPN antigen than

[0101] In some embodiments, the antibody that binds to 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).

[0102] "Full length antibody," "intact antibody" or "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0103] "Antibody fragment" refers to 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 one-armed antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0104] 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.

[0105] "Variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an 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 (FRs) and three hypervariable regions (CDRs) (see, e.g., Kindt et al., Ruby Immunology, 6th edition, WH Freeman and Co., p. 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 (see, e.g., Portolano et al., J. Immunol., 150: 880-887 (1993); Clarkson et al., Nature, 352: 624-628 (1991)).

[0106] As used herein, "hypervariable region" or "CDR" refers to each region of an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop ("hypervariable loop"). 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).

[0107] As used herein, "complementarity determining region" or "CDR" refers to the region within the CDR of the variable domain with the highest sequence variability and / or involved in antigen recognition. Typically, a natural antibody comprises 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. August 20, 1987; 196(4):901-17 and Nature. December 21-28, 1989; 342(6252):877-83.) and Lefranc et al. (IMGT-Nucleic Acids Res. January 1, 1999; 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 alternative defined CDR sequences, which may contain 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;

[0108] 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.

[0109] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (CDR) residues. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, CDR and FR sequences usually appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0110] "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From the N-terminus to the 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 the N-terminus to the 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 chain of an antibody can be divided into one of two types, referred to as kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0111] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., variant antibodies contain naturally occurring mutations or mutations that occur during the production of monoclonal antibodies, and are generally present in small amounts). Contrary to polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the term "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibody to be used can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0112] A "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0113] "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 usually 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 may 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.

[0114] "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 a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0115] "People's common 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).

[0116] As used herein, "acceptor human framework" is a framework comprising an 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. The acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence, or it may 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 a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0117] "Fc region" refers to a dimer complex comprising a 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 may include a native or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence is generally defined as extending from about position Cys226 or about position Pro230 to the carboxyl end 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 a recombinant antibody. The Fc sequence of an immunoglobulin generally includes two constant domains, a CH2 domain and a CH3 domain, and optionally includes a CH4 domain.

[0118] "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 an IgG antibody (gamma receptor), and includes receptors of FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), 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 (see, e.g., Daeron, Annu. Rev. Immunol., 15: 203-234 (1997)). 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., 1 17:587 (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).

[0119] As used herein, a "multivalent antibody" is an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have three or more antigen binding sites, and is typically not a native sequence IgM or IgA antibody.

[0120] A "multispecific antibody" is an antibody with at least two different binding sites, each with a different binding specificity. The 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.

[0121] "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.

[0122] "Fab fragment" refers to an antibody fragment containing the variable domain and constant domain of the light chain and the variable domain and first constant domain (CHI) of the heavy chain. "F(ab')2 fragment" comprises a pair of Fab fragments, which are usually covalently linked by hinge cysteines between them near their carboxyl termini. Other chemical couplings of antibody fragments are also known in the art.

[0123] As used herein, "antigen binding arm" refers to a 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.

[0124] "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 also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired antigen-binding structure.

[0125] "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 generate two antigen-binding sites.

[0126] "Linear antibodies" refer to 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.

[0127] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel.

[0128] "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.

[0129] "Specifically binds" or "specific binding" means that the antibody binds to the antigen with an affinity value of no more than about 1×10-7M.

[0130] An "affinity matured" antibody is one with one or more alterations in one or more CDRs compared to a parent antibody without such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

[0131] A "functional antigen binding site" of an antibody is a site 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 binding to antigen.

[0132] "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). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B Analyt. Technol Biomed Life Sci, 848: 79-87.

[0133] Composition or preparation

[0134] The present invention provides a composition or preparation that can be used to treat bone fractures and / or promote fracture healing, the composition or preparation 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.

[0135] 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 selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (eg, through clinical trials).

[0136] 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.

[0137] As used herein, the term "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0138] The term "physiologically acceptable carrier" refers to a carrier for active ingredients, 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 ), 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.

[0139] The pharmaceutical composition of the present invention contains 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. Usually, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0140] The effective amount of the active ingredient of the present invention may vary with 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 by the patient, the patient's body 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, satisfactory results can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally.

[0141] In the present invention, the dosage form of the composition or preparation includes (but is not limited to): solid preparation, liquid preparation, gel preparation, semi-solid preparation. Some preferred dosage forms include ointment, cream, patch, paint, spray, micro-injection, solution, etc.

[0142] 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.

[0143] 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).

[0144] Compositions for parenteral injection may include 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.

[0145] Combination medicines for inhalation may include aerosols, sprays, and the like.

[0146] The composition of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0147] 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.

[0148] The main advantages of the present invention include:

[0149] (a) It can significantly reduce the swelling and pain at the fracture ends;

[0150] (b) It can significantly increase the BV / TV (bone volume fraction) and Tb.Th (trabecular thickness) of callus tissue;

[0151] (c) It can significantly increase the maximum stress and yield stress at the fracture healing site.

[0152] 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 where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0153] Example

[0154] 1 Materials and methods

[0155] 1.1 Experimental animals

[0156] A total of 40 6-8 week old SPF male C57BL / 6 mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., animal qualification certificate number: SCXK (Shanghai) 2018-0004, and were raised in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The temperature and humidity of the breeding room were controlled at 23°C and 60%, respectively, to ensure sufficient sunlight, mouse food and drinking water. This study was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine, and the ethics approval number is PZSHUTCM211101023.

[0157] 1.2 Experimental drugs and preparation methods

[0158] Anti-mouse Podoplanin (BioXcell, BE0236, USA), Polyclonal Syrian hamster IgG (BioXcell, BP0087, USA), isoflurane (Voraid, China).

[0159] Preparation of 1mg / ml IgG isotype antibody: Dissolve 0.2ml containing 1.7mg IgG protein in 1500ul sterile PBS solution.

[0160] Preparation of 1mg / ml Anti-PDPN antibody: Dissolve 0.7ml containing 1.5mg IgG protein in 800ul sterile PBS solution.

[0161] 1.3 Experimental equipment

[0162] vivaCT40 mouse Micro-CT scanner (Scanco Medical AG, Switzerland); multi-channel small animal gas anesthesia machine (Matrx VMR, USA), micro-injector (HAMILTON, China), 1 ml sterile syringe (0.45×16 mm, Shandong Weigao), ElectroForce 3200 biomechanics machine (TA-Bose, USA), pain threshold machine (Ugo Basile, Italy), and homemade soft tissue swelling meter.

[0163] 1.4 Animal grouping and modeling

[0164] The mice were randomly divided into IgG group and Anti-PDPN group after being marked by random number table method, with 20 mice in each group. Intervention was performed 2 days before modeling. IgG group: 25ul / time IgG isotype antibody was injected locally around the popliteal lymph nodes under B-ultrasound guidance 2 days before modeling. Injection was performed once every 2 days for 4 consecutive times. Anti-PDPN group: 25ul / time anti-Podolanin antibody was injected locally around the popliteal lymph nodes under B-ultrasound guidance 2 days before modeling. Injection was performed once every 2 days for 4 consecutive times.

[0165] Right tibial fracture composite intramedullary fixation modeling method: After the mouse was anesthetized with isoflurane, it was placed in a supine position. A longitudinal incision of about 1.5 cm was made below the midpoint of the right knee of the mouse to expose the lateral edge of the middle section of the tibia, and the muscles around the tibia were bluntly separated to avoid damaging the hind limb return lymphatic vessels and lymph nodes. The 1 ml sterile syringe needle was inserted from the midpoint of the tibial plateau, and pierced into the medullary cavity about 1.5 cm along the long axis of the tibia. The needle was withdrawn to 0.2 cm proximal to the tibial plateau, and the middle section of the tibia was gently cut with a blade. After the fracture ends were aligned, the needle was inserted downward into the medullary cavity for intramedullary fixation. When the intramedullary needle was in close contact with the inner wall of the bone medullary cavity, the intramedullary needle was gently rotated clockwise or counterclockwise. It was observed that the ankle joint rotated in the same direction, which proved that the alignment was good and the modeling was successful. Cut off the needle cap exposed on the tibial plateau, suture the skin and apply antibacterial ointment to prevent infection.

[0166] 1.5 Detection indicators

[0167] 1.5.1 Improved limb swelling meter to detect soft tissue swelling at fracture site Before modeling and on the 1st, 2nd, 3rd, 5th and 7th day after successful modeling, the right hind limb of the mouse below the knee joint was completely immersed in a new practical swelling meter. After three measurements, the average value was recorded as the experimental result. The swelling degree of soft tissue at fracture site was calculated as the swelling volume after modeling / the swelling volume before modeling*100%.

[0168] 1.5.2 Plantar thermal pain threshold detection On the second day after modeling, the mice that had been fasted and deprived of water were placed in the closed chamber of the measuring instrument for 2 hours to adapt and calm down, and then 30% radiant heat was used to induce the mouse to retract its paw. When the mouse felt pain and raised its hind paw, the instrument automatically recorded the corresponding reaction time when the mouse's paw retracted. Each mouse was tested five times. After removing the highest and lowest values ​​of the five measurements, the average of the remaining three values ​​was the average reaction time of each mouse.

[0169] 1.5.3 Micro-CT scanning The mouse tibia tissue specimens treated with 75% ethanol were placed in the Micro-CT instrument detection chamber for X-ray scanning to observe the tibial fracture healing and callus formation of each group of mice. Subsequently, the tibia samples (the area between the tibial plateau and the ankle joint) were subjected to tomographic imaging with the following detection parameters: voltage 70 kV, current 142 μA, 9 μm resolution, and 0.5 mm aluminum filter. The supporting software was used to reconstruct the morphological structure of bone tissue in three dimensions, and the trabecular parameters of the callus were quantitatively analyzed.

[0170] 1.5.4 Three-point bending test After sampling, carefully remove all attached muscles from the tibia specimens, wrap them with gauze soaked in saline solution and store them at 4°C. After all specimens are collected, place them flat in the mechanical analysis machine one by one, keep the callus at the same angle and place them at the midpoint of the tester, lock both ends of the specimens, open the computer setting program, and conduct experimental analysis.

[0171] 1.6 Statistical analysis:

[0172] The statistical results were all quantitative data, described by mean ± standard deviation (±s), and analyzed using SPSS 20.0. If the two independent data groups obeyed the normal distribution and had equal variances, the t test was used; if the variances were unequal, the t' test was used. If the two independent data groups did not obey the normal distribution, the nonparametric Mann-Whitney U test was used. Repeated measures analysis of variance and simple effect test were used for the two groups of multiple time point measurement data. The test level α = 0.05, P < 0.05 represents statistical significance.

[0173] 2 Results

[0174] Figure 1 As shown in the figure, on the second day after fracture, the IgG group had obvious soft tissue swelling in the tibia and ankle joint on the fracture side, while the Anti-PDPN group had only slight soft tissue swelling in the tibia. The soft tissue swelling of the fractured part of the mice was measured by a modified limb swelling meter. On the 1st, 2nd, 3rd, 5th and 7th days after modeling, the soft tissue swelling of the fractured hind limbs in the Anti-PDPN group was significantly lower than that in the solvent group (P<0.05).

[0175] Figure 2The plantar thermal pain threshold 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).

[0176] Figure 3 The tibial callus tissue of mice 14 days after fracture was reconstructed and analyzed by Micro-CT. The Anti-PDPN group mice had large callus formation at the fracture site and the fracture line was blurred, while the IgG group mice had smaller callus formation at the fracture site and the fracture line was clear. The bone mass parameters of the tibial callus tissue of each group 14 days after fracture showed that compared with IgG, the BV / TV and Tb.Th of the callus tissue of the Anti-PDPN group mice were significantly increased (P<0.01), but there was no significant difference in Tb.N and Tb.Sp between the two groups (P>0.05). This shows that the morphometrics of the tibial callus in the Anti-PDPN group are better than those in the solvent group.

[0177] Figure 4 Biomechanical tests were 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 VEH group (P<0.05), but there was no significant difference in fracture energy (P>0.05).

[0178] The results show that Anti-PDPN can reduce swelling and pain after fracture and promote fracture healing.

[0179] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of an E11 / gp38 / podoplanin / PDPN pathway inhibitor, characterized in that: Used for preparing a composition or preparation for treating bone fractures and / or promoting bone fracture healing.

2. A composition or preparation for treating fractures and / or promoting 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 inhibitor of the E11 / gp38 / podoplanin / PDPN pathway.

3. The composition or preparation according to claim 2, 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.

4. The composition or preparation according to claim 2, characterized in that The dosage form of the composition or preparation is a topical dosage form.

5. The composition or preparation according to claim 2, characterized in that The dosage forms of the composition or preparation include ointment, cream, patch, paint, spray, microneedle and the like.

6. The composition or preparation according to claim 2, 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.

7. 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 levels of the E11 / gp38 / podoplanin / PDPN pathway in samples to which the substance to be tested has been added and samples to which the negative control has been added; if the levels of the E11 / gp38 / podoplanin / PDPN pathway in samples to which the substance to be tested has been added are reduced compared to samples to which the negative control has been added, the substance is a potential substance for treating fractures and / or promoting fracture healing.

8. The method according to claim 7, characterized in that: The method is non-diagnostic and non-therapeutic.

9. The method according to claim 7, 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.

10. The method according to claim 7, characterized in that: The detection system is a cell system.

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