Application of gefitinib in preparation of medicine for treating or preventing keloid

Abstract

The invention discloses an application of gefitinib in preparation of a medicine for treating or preventing keloid, in-vitro experiments and animal experiments show that gefitinib can block and activate cell proliferation and migration, inhibit cells from excessively synthesizing collagen and resist fibrosis. In the treatment of keloids, the supplement of gefitinib can effectively reduce the fibroblast proliferation of keloids and inhibit cell migration and extracellular matrix synthesis no matter in a cell experiment or an animal experiment, so that fibrosis is relieved, key cell activities causing the formation and development of keloids are effectively suppressed, and the curative effect of keloids is improved. Powerful support is provided for treating keloids from the cellular level. The experimental result of constructing a humanized keloid mouse model shows that the gefitinib remarkably relieves the fibrosis level of keloid.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of gefitinib in preparing a medicine for treating or preventing keloid. Background Art

[0002] Keloid, as a benign fibroproliferative skin tumor, has unique pathological manifestations. During the healing process of normal skin wounds, the proliferation of fibroblasts and the deposition of extracellular matrix are in an orderly and controllable state. However, the fibroblasts in patients with keloids show abnormal proliferation and excessive deposition of extracellular matrix. This abnormality usually occurs in genetically susceptible individuals and is mostly caused by skin injuries such as burns, surgical incisions, and acne. The scar tissue formed will not only gradually increase over time, but also exceed the original wound boundaries and lack the ability to disappear on its own. In appearance, keloids appear as raised, hard lumps, often red or purple in color, which cause obvious appearance deformity problems to patients.

[0003] At present, the incidence of keloids in the population cannot be underestimated, and different races and age groups may develop the disease. Many factors are involved in its occurrence and development. Genetic factors lay the foundation for the formation of keloids, and individuals carrying related genetic mutations or polymorphisms are more susceptible to the disease. The tension at the site of skin injury affects the probability of keloid formation, and high-tension areas such as shoulders and chests are more susceptible to the disease. Excessive inflammatory response and immune abnormalities also play an important role in the pathogenesis of keloids. The continuous release of inflammatory mediators and immune cell dysfunction may promote the formation and progression of keloids. However, despite its high incidence, the exact pathological mechanism has not yet been fully clarified, which greatly limits the formulation of effective clinical prevention and treatment strategies.

[0004] Surgical excision is one of the common treatments for keloids, but the recurrence rate after surgical treatment alone is extremely high and may even aggravate the symptoms of keloids. In clinical practice, 5-fluorouracil combined with glucocorticoids is often used for non-surgical treatment. 5-fluorouracil can inhibit cell proliferation, while glucocorticoids can reduce inflammatory responses and inhibit fibroblast activity. However, the efficacy of this combined treatment is limited, and the recurrence rate remains high. On the one hand, some patients do not respond well to drugs and cannot effectively control the growth of keloids; on the other hand, long-term use of such drugs may cause adverse reactions such as skin atrophy and pigmentation. Therefore, the development of safe and effective drugs for the treatment of keloids has become an important issue that needs to be urgently addressed in the current medical field. Summary of the invention

[0005] The main purpose of the present invention is to propose an application of gefitinib in the preparation of a drug for treating or preventing keloids, aiming to provide a safe and effective drug for treating keloids, which can effectively reduce the proliferation of keloid fibroblasts, inhibit cell migration and extracellular matrix synthesis, and thus reduce fibrosis.

[0006] To achieve the above object, the present invention proposes the use of gefitinib in preparing a drug for treating or preventing keloid.

[0007] Keloid is a difficult-to-treat disease that affects human health. Its specific biological mechanism has not been fully elucidated, so the development of effective drug treatments is still a huge challenge. Fibroblasts can synthesize collagen and remodel the extracellular matrix, and are considered to be the main effector cell type involved in skin fibrosis. However, there has been a lack of in-depth and accurate research on how they specifically affect and ultimately lead to the biological mechanism of fibrosis.

[0008] Preferably, the gefitinib achieves therapeutic effect by inhibiting fibrosis of scar tissue.

[0009] Preferably, the gefitinib treats or prevents keloids by inhibiting cell proliferation, cell migration, and extracellular matrix synthesis.

[0010] Preferably, the gefitinib inhibits the EGFR-MAPK pathway, thereby preventing the transformation of fibroblasts in keloids into myofibroblasts and alleviating skin fibrosis.

[0011] As a frontline clinician and medical researcher, the inventor started from clinical problems and revealed, found and verified the new specific mechanism by which AREG aggravates keloids through cell and animal experiments. He first proposed that AREG activates the EGFR-MAPK pathway, induces the transformation of fibroblasts in keloids into myofibroblasts, and promotes the development of keloids.

[0012] Preferably, the drug is a topical preparation selected from an injection, a sustained-release gel, a transdermal patch or an external cream.

[0013] Preferably, the administration method of gefitinib is intralesional injection, with a dosage of 50-200 mg / kg body weight.

[0014] Preferably, the dosage of gefitinib is 100 mg / kg body weight.

[0015] Preferably, the drug comprises gefitinib and excipients.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) In the technical solution provided by the present invention, the inventors, as frontline clinicians and medical researchers, based on clinical problems, revealed a new specific mechanism by which AREG aggravates keloids through cell and animal experiments, namely, AREG activates the EGFR-MAPK pathway, induces the transformation of fibroblasts in keloids into myofibroblasts, and promotes the development of keloids. This provides a new perspective for understanding the pathogenesis of keloids. Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Based on this mechanism, the research has a clear target of action and intervenes in the development of keloids from the root. (2) The present invention has shown through in vitro experiments and animal experiments that gefitinib can block activated cell proliferation and migration, inhibit excessive cell synthesis of collagen and resist fibrosis. In the treatment of keloids, whether in cell experiments or animal experiments, supplementation with gefitinib can effectively reduce the proliferation of keloid fibroblasts, inhibit cell migration and extracellular matrix synthesis, and thus reduce fibrosis, effectively curbing the key cell activities that lead to the formation and development of keloids, and providing strong support for the treatment of keloids at the cellular level. The experimental results of the present invention through the construction of a humanized keloid mouse model showed that gefitinib significantly reduced the fibrosis level of keloids. The experimental results of this animal model provide important preliminary data support for the application of gefitinib in the treatment of human keloids, and prove its effectiveness in the treatment of keloids in vivo.

[0017] (3) Gefitinib used in the present invention is a widely used marketed drug in clinical practice, with sufficient research on its pharmacokinetics and safety, and relatively low toxic and side effects. Compared with the development of new drugs with unknown safety for the treatment of keloids, this greatly reduces the risk of clinical application, is more easily accepted by patients, and reduces the additional burden caused by adverse drug reactions during treatment. This scheme applies gefitinib to the treatment of keloids for the first time, providing a new strategy for the drug treatment of keloids and broadening the scope of application of existing treatment methods. In clinical practice, it provides new possibilities for solving the difficult problem of keloid treatment, and is expected to change the current situation of keloid being difficult to treat, and bring new treatment options to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1This is a diagram showing the effect of gefitinib on the activity of keloid fibroblasts detected by the CCK8 experiment in Example 1 provided by the present invention; Figure 2 This is a diagram showing the effect of gefitinib on the migration ability of keloid fibroblasts detected by the cell migration experiment in Example 1 provided by the present invention, with a scale of 50 μm; Figure 3 The mRNA level of Collagen I and Collagen III detected by the fluorescence quantitative PCR technology in Example 1 provided by the present invention after intervention with different concentrations of gefitinib; Figure 4 The results of Western blot detection of Collagen I, Collagen III and GAPDH expression after intervention with different concentrations of gefitinib in Example 1 provided by the present invention; Figure 5 This is a Reactome enrichment analysis diagram of differentially down-regulated genes between the gefitinib intervention group and the control group in Example 1 provided by the present invention; Figure 6 This is a GSEA (Gene Set Enrichment Analysis) enrichment analysis diagram of differentially expressed genes between the gefitinib intervention group and the control group in Example 1 provided by the present invention; Figure 7 This is a comparison diagram of the general observation of the keloid nodules in nude mice in Example 2 provided by the present invention; Figure 8 This is a comparison diagram of the volume of keloid tissue blocks in nude mice in Example 2 provided by the present invention; Fig. 9 This is a comparison diagram of the mass of nude mouse keloid tissue blocks in Example 2 provided by the present invention; Fig.10 This is a Masson staining result of the keloid tissue block of nude mice after treatment with gefitinib in Example 2 provided by the present invention, the scale is 50 μm, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001.

[0020] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0021] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0022] Example 1 In vitro experimental study on the inhibition of proliferation, migration and extracellular matrix synthesis of keloid fibroblasts by gefitinib (1) Isolation and culture of primary keloid fibroblasts After the patient agreed and signed the informed consent form, the tissue removed from the keloid patient after surgery was brought to the laboratory to extract primary fibroblasts. First, the excised keloid tissue was placed in sterile saline containing penicillin-streptomycin to prevent bacterial infection. Subsequently, the tissue sample was minced and digested with collagenase (4 mg / mL) at 37°C for 6 hours. After centrifugation, the cell pellet was collected and resuspended in DMEM medium containing 10% fetal bovine serum (FBS), inoculated in a culture dish, and cultured in an incubator at 37°C and 5% CO2. When the cells grew to 80% confluence, they were subcultured and the 3rd to 6th generation cells were taken for subsequent experiments.

[0023] (2) Gefitinib inhibits the proliferation activity of keloid fibroblasts Keloid fibroblasts were seeded in 96-well plates (5 × 10 3 cells / well), the culture medium was 200 μl DMEM containing 10% fetal bovine serum. After overnight culture, the cell viability was detected using the CCK8 kit according to the instructions, and the optical density at 450 nm was measured at 24 and 48 hours, respectively. The results are shown in Figure 1 As shown. Figure 1 It can be seen that compared with the optical density value of the control group, the cell viability decreased after gefitinib treatment, and as the drug concentration increased, the inhibition of cell activity was dose-dependent.

[0024] (3) Gefitinib inhibits the migration of keloid fibroblasts When the cell density of keloid fibroblasts reached more than 90%, a 200 μL pipette tip was used to make uniform scratches on the cell monolayer, and the cells were then placed in serum-free medium with or without gefitinib for 48 hours.

[0025] Images were collected at 0, 24, and 48 hours after scratching. Figure 2 The experimental results are based on the measurements of scratch areas in 6 randomly selected fields of view in each well, and the experiment was repeated using 6 independent cell sample pools to ensure data reliability. Figure 2 The results showed that gefitinib can inhibit the migration of keloid fibroblasts.

[0026] (4) Gefitinib inhibits extracellular matrix synthesis in keloid fibroblasts First, the real-time quantitative PCR method was used to detect the changes in mRNA. Figure 3 As shown. Total RNA of cells was extracted using Trizol reagent (Invitrogen) according to the instructions. The first-strand cDNA was synthesized using PrimeScript RT kit (Takara, Takara Biotech Co., Ltd., Tokyo, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the endogenous standard. Quantitative real-time PCR was performed using SYBR Green Master Mix according to the instructions. The reaction conditions were: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles (denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds). The Ct value (threshold cycle number) of each sample was obtained by calculation, and the relative mRNA expression was normalized based on the β-actin value. Combined with Figure 3 Results Through analysis, we found that gefitinib inhibited the synthesis of type I and type III collagen in keloid fibroblasts.

[0027] Subsequently, protein level changes were detected by Western blotting. Figure 4 As shown. The cells were lysed in 1× SDS-PAGE loading buffer (containing 50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% BPB, 10% glycerol, and 50 mM β-glycerophosphate). The lysate was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane for Western blot detection. The signal was detected using an enhanced chemiluminescence detection kit. Figure 4 The results showed that, consistent with the changes in mRNA, gefitinib inhibited the synthesis of type I and type III collagen in keloid fibroblasts in a concentration gradient-dependent manner.

[0028] (5) Transcriptome analysis of keloid fibroblasts treated with gefitinib and enriched pathways In order to further explore the mechanism of action of gefitinib on keloid, we performed RNA sequencing and analysis on cells treated with gefitinib. Total RNA was extracted from cells using TRIzol reagent (Takara). RNA sequencing was performed by BGI. Differential expression analysis was performed on fibroblasts with and without gefitinib stimulation using DESeq2 software, with a false discovery rate (FDR) <0.5 and a log2 fold change >0.58 as the screening threshold. For the analysis of differentially expressed genes in three groups of normal skin fibroblasts (transfected with Fn14 siRNA or control siRNA, respectively), the log2 fold change >0.58 was set as the screening threshold. We performed Reactome enrichment analysis on the differentially expressed genes, and the results are shown in the figure below. Figure 5 As shown by Figure 5 It was found that the down-regulated differentially expressed genes were enriched in pathways related to extracellular matrix synthesis, such as Extracellular matrix organization, Collagen biosynthesis and modifying enzymes, Collagen chain trimerization, Collagen formation, Assembly of collagen fibrils and other multimeric structures, and ECM proteoglycans, proving that gefitinib inhibits extracellular matrix synthesis.

[0029] pass Figure 6 The GSEA (Gene Set Enrichment Analysis) enrichment analysis of differentially expressed genes found that the MAPK_FAMILY_SIGNALING_CASCADES signaling pathway was significantly enriched in the lower part of the gene ranking list (NES=-1.62, p<0.05), indicating that the genes related to this pathway showed an overall down-regulation trend in the gefitinib treatment group. This finding is consistent with existing research reports on gefitinib in the treatment of diseases such as non-small cell lung cancer (NSCLC).

[0030] Example 2 Animal Experimental Study on the Effect of Gefitinib on Reducing the Degree of Keloid Fibrosis (1) Establishment of humanized keloid mouse model and drug administration After the patients agreed and signed the informed consent form, skin lesion tissue was obtained from the keloid patients after surgery. The excised keloid tissue was placed in sterile saline containing antibiotics to prevent bacterial infection, and then centrifuged, washed and cut to remove the epidermis of the keloid tissue and cut into tissue blocks of uniform size (5×4×3mm).

[0031] Immunodeficient nude mice were selected and anesthetized with 1% sodium pentobarbital. After anesthesia, a surgical incision was prepared under the skin of the mouse's back. The surgical area was cleaned and disinfected, and the treated keloid tissue was transplanted into the mouse's subcutaneous tissue. The skin incision was sutured with a fine needle and sutures to construct a humanized keloid mouse model. One week after modeling, keloid-like nodules were observed on the back of the modeled mice, indicating that the modeling was successful. DMSO was given to the five mice with successful modeling, and 100 mg / kg gefitinib was injected intralesionally in the other five mice with successful modeling. The injections were continued for three weeks, and the keloid nodules were observed by taking pictures. The results are as follows: Figure 7 shown.

[0032] (2) Observation and comparison of keloid fibrosis We established a humanized keloid mouse model and administered the drug, and then conducted regular observations and measurements to measure the volume of keloid tissue in nude mice. Figure 8 ; Measure the mass of nude mouse keloid tissue blocks and obtain Fig. 9 All the results were summarized and the growth curve of keloid nodules was drawn. Figure 8 and 9 It was found that gefitinib inhibited the growth of keloid nodules in nude mice. In addition, the volume and mass of keloids in nude mice also showed the same trend. The mice given gefitinib had smaller nodules. Fibrosis is characterized by excessive deposition of extracellular matrix (especially collagen fibers). Masson staining results of keloid tissue blocks of nude mice injected with gefitinib and without gefitinib were obtained. Fig.10 Masson staining can visually display the proliferation of collagen fibers, thus helping to determine the degree of fibrosis. Fig.10 The results of Masson staining showed that compared with the control group, the group treated with 100 mg / kg gefitinib in the skin lesions had a significantly reduced fibrosis level after 3 weeks compared with the model mice that were not injected with gefitinib.

[0033] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. The use of gefitinib in the preparation of drugs for treating or preventing keloid.

2. The use according to claim 1, characterized in that The gefitinib achieves its therapeutic effect by inhibiting the fibrosis of scar tissue.

3. The use according to claim 1, characterized in that The gefitinib achieves the treatment or prevention of keloid by inhibiting cell proliferation, cell migration and extracellular matrix synthesis.

4. The use according to claim 1, characterized in that The gefitinib inhibits the EGFR-MAPK pathway, thereby preventing the transformation of fibroblasts in keloids into myofibroblasts and alleviating skin fibrosis.

5. The use according to claim 1, characterized in that The drug is a topical preparation, which is selected from injections, sustained-release gels, transdermal patches or external creams.

6. The use according to claim 1, characterized in that The administration method of gefitinib is intralesional injection, with a dosage of 50-200 mg / kg body weight.

7. The use according to claim 6, characterized in that The dosage of gefitinib is 100 mg / kg body weight.

8. The use according to claim 1, characterized in that The medicine comprises gefitinib and excipients.

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