Application of TrkA inhibitor in treatment of tendon adhesion
The TrkA inhibitor TPX-0005 inhibits the TrkA signaling pathway, and solves the side effects and unsatisfactory effects of existing treatments for tendon adhesions, achieving the effect of reducing tendon adhesions and improving tendon function.
Patent Information
- Application Number
- CN202510446932.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-08-08
AI Technical Summary
Existing treatments for tendon adhesions such as surgery, drugs and biological materials have side effects or are not effective, making it difficult to effectively alleviate tendon adhesions and improve tendon function.
The TrkA inhibitor TPX-0005 is used to inhibit the TrkA signaling pathway, interfere with the mechanism of tendon adhesion, reduce the abnormal differentiation of mesenchymal stem cells into myofibroblasts, and local injection is used to treat tendon adhesions.
Effectively reduce tendon adhesions, improve tendon biomechanical properties, and show good clinical conversion potential at safe doses.
Smart Images
Figure CN120437299A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to application of a TrkA inhibitor in treating tendon adhesion, and belongs to the technical field of biomedicine. Background Art
[0002] Tendon adhesions are a complex pathological condition characterized by excessive scarring. Its pathological basis is the excessive differentiation of mesenchymal stem cells into myofibroblasts, which become the primary driver of adhesions. This is manifested by increased collagen deposition, restricted range of motion, and decreased tissue function. Hydrogels are physical barriers used to prevent or limit tendon adhesions. Loading drugs onto hydrogels to inhibit the growth of adhesion tissue is a common strategy. While biological barrier materials offer physical barrier properties, they can also cause foreign body reactions that can aggravate adhesions. Drugs used to treat tendon adhesions, such as anti-inflammatory drugs (such as celecoxib), antioxidants (such as hydrogen-rich water), and antimetabolites (such as angiotensin-converting enzyme inhibitors and rapamycin), either have unsatisfactory anti-adhesion effects or have side effects, such as impaired tendon healing. Surgical excision of adhesion tissue can lead to re-adhesion, infection, and loss of function, while physical therapy can cause pain and has limited effectiveness in severe cases.
[0003] TPX-0005 (trade name Repotrectinib) is a new-generation, multi-targeted tyrosine kinase inhibitor (TKI) specifically designed to inhibit molecular targets such as TrkA / B / C, ROS1, and ALK. It was initially developed for the treatment of solid tumors carrying fusions or mutations in these genes, such as lung cancer and other ROS1-, NTRK-, and ALK-positive tumors. In anti-tumor therapy, TPX-0005 has attracted attention for its potent inhibitory ability against "solvent-front" mutations, which often lead to drug resistance.
[0004] Traditional treatments, such as surgery, often lead to repeated injuries and induce re-adhesion. Small molecule drugs, such as aspirin, often cause severe gastrointestinal bleeding and biomaterial rejection. In short, existing treatments and their efficacy are limited in alleviating adhesions. Summary of the Invention
[0005] The purpose of the present invention is to provide a TrkA inhibitor for the treatment of tendon adhesions. The present invention verifies through experiments that TPX-0005 intervenes in the pathogenesis of tendon adhesions by inhibiting the TrkA signaling pathway, thereby reducing the abnormal differentiation of mesenchymal stem cells into myofibroblasts from the source, thereby reducing the degree of adhesion, and improving tendon repair function to a certain extent, thereby promoting functional repair of damaged tendons.
[0006] To achieve the above objectives, the present invention provides the use of a TrkA inhibitor in the preparation of a medicament for preventing and / or treating tendon adhesions. The TrkA inhibitor is a substance that inhibits or reduces the expression or activity of the TrkA gene and / or protein.
[0007] Preferably, the TrkA inhibitor comprises at least one of a knockdown agent, a knockout agent for the TrkA gene, or a receptor blocking agent for the TrkA protein.
[0008] Preferably, the knockdown agent for the TrkA gene comprises any one of shRNA, siRNA, vector or CRISPR / Cas system reagents targeting the TrkA gene.
[0009] Preferably, the knockout agent for the TrkA gene is a reagent comprising a Cre / LoxP system, which is used to specifically knock out the TrkA gene in mesenchymal hepatocytes. The TrkA gene is specifically knocked out by driving CreERT2 via the Prrx1 promoter.
[0010] Preferably, the receptor blocking agent comprises a small molecule compound or a polypeptide targeting the TrkA protein.
[0011] Preferably, the TrkA inhibitor comprises TPX-0005.
[0012] Preferably, the vector comprises any one of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, and a liposome.
[0013] Preferably, the drug is a drug targeting TrkA gene or protein in mesenchymal stem cells.
[0014] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient is a TrkA inhibitor.
[0015] Preferably, the dosage form of the drug includes an injection, and the administration method is local injection.
[0016] The chemical structure of TPX-0005 is shown below:
[0017]
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1. The present invention knocks out Prrx1 by constructing TrkAA mouse tendon injury model was developed, demonstrating that the TrkA signaling pathway promotes tendon adhesions. Furthermore, local injection of the TrkA inhibitor TPX-0005 into mice with tendon adhesions demonstrated that TPX-0005 can effectively reduce tendon adhesions and improve tendon biomechanical properties in the long term.
[0020] 2. By detecting ROS, apoptosis, cell cycle, and mitochondrial membrane potential in local tissues, the present invention demonstrates that TPX-0005 is safe at the experimental dose and has minimal off-target effects. Therefore, the TPX-0005 provided by the present invention has good application prospects and clinical translation potential as a drug for the treatment of tendon adhesions. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Results of TrkA signaling pathway promoting tendon adhesion: (A) gross scoring results, (B) HE and Masson staining results, (C) αSMA immunofluorescence staining results;
[0022] Figure 2 The therapeutic effect of TPX-0005 on tendon adhesion in mice: (A) gross score, HE and Masson staining results, (B) HE staining statistical graph, (C) gross score statistical graph, (D) αSMA immunofluorescence staining results, (E) biomechanical tendon maximum load test results, (F) biomechanical tendon stiffness test results;
[0023] Figure 3 Safety testing of TPX-0005: (A) ROS detection results, (B) cell cycle detection results, (C) cell apoptosis detection results, and (D) mitochondrial membrane potential detection results. DETAILED DESCRIPTION
[0024] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.
[0025] It should be understood that the terms used herein are intended only to describe particular embodiments and are not intended to limit the present invention. Furthermore, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range, within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range are also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded within the range.
[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.
[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0029] Unless otherwise specified, the biological materials or reagents used in the following examples of the present invention are all commercially available products and can be purchased through conventional channels.
[0030] Example
[0031] In this example, C57BL / 6J mice were purchased from the Animal Laboratory of Shanghai Sixth People's Hospital and used as wild-type mice (WT); Prrx1-CreERT2 mice and Rosa26-TrkA-Loxp mice were purchased from Jicui Pharmaceutical Company and used to construct mesenchymal stem cell-specific TrkA receptor knockout mice (Prrx1 TrkA ) and was constructed by the company using Cre-LoxP conditional gene editing technology to construct homozygous Prrx1-CreERT2::Rosa26-TrkA-Loxp mice, which are TrkA receptor knockout mice (abbreviated as Prrx1 TrkA This experiment was approved by the Ethics Committee of Shanghai Sixth People's Hospital.
[0032] The transgenic mice were divided into two groups: one group injected with sterile saline and the other group injected with an equal amount of tamoxifen (TMX). TMX can induce the activation of Cre enzyme to knock out the TrkA receptor. A tendon adhesion model was then established.
[0033] Tendon Adhesion Model: The flexor digitorum profundus tendon was sectioned and Kessel sutured. After anesthetizing the mouse, the skin of the right hind paw was disinfected with 75% ethanol. The flexor digitorum profundus tendon was exposed through a posterior midline incision and transected laterally. The tendon was then repaired using a modified Kessel pattern to create a tendon adhesion model. The wound was closed with 5-0 sutures.
[0034] After modeling, all mice received the same treatment and were allowed to move freely in the cage. After 14 days and 28 days, the mice were euthanized by inhalation of carbon dioxide and then perfused with 4% paraformaldehyde through the femoral artery for 5 minutes. The right foot was then removed, immersed in 4% paraformaldehyde for 24 hours, and decalcified in 0.5M ethylenediaminetetraacetic acid (VWR, 0105) (pH 7.5) for one week. Finally, the specimens were dehydrated with 30% sucrose (Sigma-Aldrich, S9378) at 4°C and embedded in the optimal cutting temperature compound (OCT) at -80°C. Gross scoring, HE & Masson staining, and immunofluorescence staining of αSMA were performed on each group of mice to verify their phenotype.
[0035] Gross ROM (Range of Motion): The hind limbs of mice in each group were amputated to the knee, and the skin at the ankle was removed. First, the tibia was fixed with a clamp. Then, the FDL (Flexor Digitorum Longus) was pulled with a 19-gram weight to measure the change in the metatarsophalangeal joint angle.
[0036] HE & Masson staining: Tendons were stained using a hematoxylin and eosin (HE) staining kit (Beyotime, C0105S) and a Masson trichrome staining kit (Beyotime, C0189S).
[0037] Immunofluorescence staining of αSMA: Before blocking nonspecific sites with 5% serum, the sections were hydrated with PBS (Solarbio, SL050), and then incubated with the primary antibody αSMA (Cell Signaling, 19245) at 4°C overnight. The secondary antibody was then incubated with the sections at room temperature for 1 hour. Finally, the sections were mounted with anti-fluorescence quenching mounting medium containing DAPI (Beyotime, P0131). Images were taken with a Zeiss 780 confocal microscope, and positive cells were quantified using ImageJ software.
[0038] The test results showed that adhesion was reduced, such as Figure 1 As shown, the overall score test results ( Figure 1 A) Compared with the Veh group, the joint mobility of mice in the TrkA receptor knockout group (TMX) increased. HE & MASSON staining results ( Figure 1 B) Compared with the Veh group, the adhesion between the tendon and the surrounding tissues of the mice in the TMX group after knocking out the TrkA receptor was reduced; T represents tendon, M represents muscle, the black dotted line represents the part where the tendon adheres to the surrounding tissues, and the yellow dotted line represents the part where the tendon does not adhere to the surrounding tissues. αSMA results ( Figure 1 C) Compared with the Veh group, the activation of mouse myofibroblasts was reduced in the TrkA receptor knockout group (TMX).
[0039] WT mice were divided into two groups: a control group (WT + Veh) and an experimental group (WT + TPX-0005). The results were divided into two time points: 14 and 28 days. C57BL / 6J mice in the WT + Veh and WT + TPX-0005 groups underwent transection of the flexor digitorum profundus tendon and Kessel suture to establish a tendon adhesion model. The WT + TPX-0005 group was then injected with TPX-0005 (Repotrectinib, purchased from MCE, HY-103022) at a concentration of 50 mg / ml in DMSO. Starting from the date of PA modeling, TPX-0005 was administered locally at the modeling site (10 μl per injection) every 7 days until specimen collection. The injection method was 10 μL per injection at the modeling site at a concentration of 20 ng / mL. The WT + Veh group was injected with 10 μL of Veh (i.e., sterile DMSO) to serve as a control group. After modeling, all mice received the same treatment and were allowed to move freely in their cages. Tendon tissue samples were collected 14 and 28 days later for gross evaluation, HE and Masson staining, and immunofluorescence staining for αSMA to verify phenotypes. Tendon biomechanics were also assessed to evaluate tendon healing.
[0040] Biomechanical testing: The maximum load and stiffness of the repaired tendons were collected on days 14 and 28 after repair using a dynamic mechanical analyzer Q800 (TA Instruments). The proximal end was stretched at a rate of 30 mm / min until fracture, and the force-displacement curves were obtained by digital calculation.
[0041] The test results showed that adhesion was reduced, such as Figure 2 As shown, the overall score test results ( Figure 2 A, 2C) showed that the joint mobility of the experimental group WT+TPX-0005 mice increased compared with WT+Veh. HE&MASSON staining results ( Figure 2 A, 2C) show that compared with WT+Veh, the adhesion between tendon and surrounding tissues in the experimental group WT+TPX-0005 mice was reduced; T represents tendon, M represents muscle, black dotted line represents the part where tendon is adhered to surrounding tissues, and yellow dotted line represents the part where tendon is not adhered to surrounding tissues. αSMA results ( Figure 2D) Compared with WT+Veh, the activation of myofibroblasts in the experimental group WT+TPX-0005 mice was reduced; there was no significant difference in biomechanics at 14 days, but it improved at 28 days ( Figure 2 EF).
[0042] The fibrotic tissues of the control group WT + Veh and the experimental group WT + TPX-0005 were collected, and the samples were minced and digested into single cells with type I collagenase (Servicebio, GC305013). Reactive oxygen species (ROS), cell apoptosis, cell cycle, and mitochondrial membrane potential were detected to evaluate their ROS, cell apoptosis, cell cycle, and mitochondrial membrane potential.
[0043] ROS Assay: Treat cells with 1 mL of 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, S0035S) diluted in serum-free medium and resuspend. Incubate cells in a 37°C incubator for 30 minutes. Then, rinse cells three times with serum-free medium. Fluorescence intensity was measured by flow cytometry, and data were analyzed using FlowJo software.
[0044] Cell cycle analysis: After harvesting, cells were washed with cold PBS and fixed in 70% ethanol at 4°C for 30 minutes. Cells were then washed again with PBS, fixed, and incubated with a PI staining solution (50 μg / mL PI and 50 μg / mL RNase) for 30 minutes at room temperature in the dark. Finally, cells were harvested and analyzed by flow cytometry, with data analyzed using FlowJo software.
[0045] Mitochondrial membrane potential (MMP) assay: Mitochondrial membrane potential was assayed using the JC-10 fluorescent probe (YEASEN, 40707ES) according to the manufacturer's instructions. After preparing the working solution, cells were harvested and incubated with 500 μL of JC-10 working solution at 37°C for 30 minutes in the dark. After rinsing the cells with PBS, the mitochondrial membrane potential was measured by flow cytometry using fluorescence intensity in the FL1 and FL2 channels.
[0046] Annexin V-FITC / PI assay for apoptosis: Resuspend cells, wash twice with PBS, and centrifuge. Resuspend cells in 195 μL of Annexin V-FITC binding buffer, add 5 μL of Annexin V-FITC and 10 μL of PI, and incubate at room temperature in the dark for 15 minutes. Subsequently, cells were harvested and analyzed using a CytoFLEX LX flow cytometer. Data were analyzed using FlowJo software.
[0047] Test results such as Figure 3As shown, there was no change in the experimental group (WT+TPX-0005) compared with the control group (WT+Veh).
[0048] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.
Claims
1. Use of a TrkA inhibitor in the preparation of a medicament for preventing and / or treating tendon adhesion.
2. The use according to claim 1, characterized in that The TrkA inhibitor includes at least one of a knockdown agent, a knockout agent for the TrkA gene, or a receptor blocking agent for the TrkA protein.
3. The use according to claim 2, characterized in that The knockdown agent for the TrkA gene includes any one of shRNA, siRNA or vector targeting the TrkA gene or a reagent of the CRISPR / Cas system.
4. The use according to claim 2, characterized in that The knockout reagent for the TrkA gene includes a reagent of the Cre / LoxP system targeting the TrkA gene, which is used to specifically knock out the TrkA gene in mesenchymal hepatocytes.
5. The use according to claim 2, characterized in that The receptor blocking agent includes a small molecule compound or a polypeptide targeting the TrkA protein.
6. The use according to claim 2, characterized in that The TrkA inhibitors include TPX-0005.
7. The use according to claim 3, characterized in that The vector includes any one of an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, and a liposome.
8. The use according to any one of claims 1 to 7, characterized in that The drug is a drug targeting the TrkA gene or protein in mesenchymal stem cells.
9. The use according to any one of claims 1 to 7, characterized in that The medicine comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is a TrkA inhibitor.
10. The use according to claim 9, characterized in that The dosage form of the drug includes injection, and the administration method is local injection.