CN115869317A - Application of erlotinib in preparation of medicine for preventing/treating subretinal membrane generation after retinal detachment - Google Patents

Abstract

The invention provides an application of erlotinib in preparing a medicine for preventing/treating subretinal membrane generation after retinal detachment. In animal experiments, 40mg/kg of erlotinib orally can effectively prevent the generation of the subretinal membrane after long-term retinal detachment, and can effectively remove the existing subretinal membrane for treatment. The integrity of the subretinal space tissue structures (retinal pigment epithelium, photoreceptor cell outer segments, etc.) was unaffected by treatment with this concentration of erlotinib. In cellular experiments, erlotinib at a concentration of 5uM was able to significantly inhibit fibronectin production by RPE cells and promote fibronectin degradation. Erlotinib can be demonstrated to prevent/or treat subretinal membrane production following retinal detachment.

Description

The effect of erlotinib on the production of subretinal membrane after the preparation of prevention/treatment of retinal detachment application in medicine

technical field

The invention belongs to the new field of medical application, and particularly relates to the application of erlotinib in the preparation of drugs for preventing/treating subretinal membrane production after retinal detachment.

Background technique

Subretinal membrane is a type of proliferative vitreoretinopathy (PVR). The occurrence of PVR is the main reason for the failure of rhegmatogenous retinal detachment reduction surgery. The pathological manifestations are the shrinkage and stretching of extensive fibrous proliferation membranes on the front and back surfaces of the retina, resulting in retinal detachment. At present, the clinical treatment for PVR is to surgically remove the proliferative membrane, but since the subretinal membrane is located in the subretinal space, its removal requires an incision and stoma to the retina, and this surgical operation will inevitably cause damage to the tissue structure of the subretinal space. irreversible damage. Therefore, the difficulty and complications of subretinal membrane surgery are greatly increased compared with epiretinal membrane (ERM). How to remove the subretinal membrane while maintaining the in-situ state of the retina has always been the ultimate goal of improving surgical methods. In addition, due to the poor understanding of the pathogenesis of the subretinal membrane, the development of drugs targeting the subretinal membrane is also difficult.

Contents of the invention

The purpose of the present invention is to provide a new application of erlotinib in the preparation of medicines, in particular to provide the application of erlotinib in the preparation of medicines for preventing/treating the production of subretinal membrane after retinal detachment.

The present invention provides a new medical application of the existing drug erlotinib. Erlotinib is a reversible tyrosine kinase inhibitor, which can inhibit the phosphorylation of intracellular tyrosine kinase related to a specific type of epidermal growth factor receptor (EGFR), which is the EGFR gene First-line therapy for patients with locally advanced or metastatic non-small cell lung cancer with sensitive mutations. But there is no report on the treatment of PVR.

This study found that single-cell transcriptome sequencing of human subretinal membrane surgical specimens showed that the main cells and extracellular matrix components of the subretinal membrane were derived from RPE cells. The matrix composition is highly similar to that of humans, and it was found that EGFR phosphorylated at Tyr1068 was specifically expressed in the subretinal membrane after surgical resection and animal modeling. In animal experiments, oral administration of 40mg/kg erlotinib can effectively prevent the generation of subretinal membrane after long-term retinal detachment, and can effectively remove the existing subretinal membrane to play a therapeutic role. Under the treatment of this concentration of erlotinib, the integrity of the tissue structure of the subretinal cavity (retinal pigment epithelial cells, outer segments of photoreceptor cells, etc.) was not affected. In cell experiments, erlotinib at a concentration of 5uM can significantly inhibit the production of fibronectin in RPE cells and promote the degradation of fibronectin. It can be confirmed that erlotinib can prevent/or treat subretinal membrane production after retinal detachment.

Description of drawings

Fig. 1 is a graph showing the proportion of different cell types in single-cell transcriptome sequencing of human subretinal membrane surgical specimens in the embodiment.

Fig. 2 is a single-cell transcriptome sequencing analysis diagram of the human subretinal membrane surgical specimen in the embodiment.

Fig. 3 is an immunofluorescence image of retinal pigment epithelial cells treated with different concentrations of erlotinib in the example.

Fig. 4 is an immunofluorescence image of retinal pigment epithelial cells treated with different concentrations of erlotinib in the example.

Fig. 5 is a real-time quantitative PCR diagram of retinal pigment epithelial cells treated with different concentrations of erlotinib in the example.

Fig. 6 is an immunofluorescence staining diagram of eyeball sections of mice with retinal detachment in the embodiment.

Fig. 7 is an immunofluorescence staining diagram of eyeball sections of mice with retinal detachment in the embodiment.

Fig. 8 is an immunofluorescence staining diagram of eyeball sections of mice with retinal detachment in the embodiment.

Detailed ways

In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiment of the application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the application. Obviously, the described embodiment is only It is an embodiment of a part of the application, but not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.

It should be noted that the terms "comprising" and "having" in the specification and claims of the present application and the above-mentioned drawings, as well as any variations thereof, are intended to cover non-exclusive inclusion, for example, including a series of steps or units A process, method, system, product or device is not necessarily limited to those steps or elements explicitly listed, but may include other steps or elements not explicitly listed or inherent to the process, method, product or device.

PCA clustering was performed on the single-cell transcriptome sequencing data of human subretinal membrane surgical specimens, and different cell clusters were defined based on the specific markers expressed by cells, and the proportion of each cell type was calculated. The obtained results are shown in Figure 1, confirming that the cell types of the subretinal membrane are mainly derived from RPE.

By enriching the pathways of normal RPE differentiation intermediate state and pathological dedifferentiation intermediate state cells to find the consistent gene set that is crucial in the differentiation/dedifferentiation process (Fig. 2 left), and extract the genes in the consistent pathway, According to the descending order of its expression ratio in the disease group, the specific expression gene Top1 in the process of dedifferentiation was screened——EGFR (Fig. 2 right). In vitro experiments:

RPE cells were treated with different concentrations (0uM, 2uM, 5uM) of EGFR inhibitor erlotinib (MCE, HY-50896) for 72 hours, and fibronectin (FN1) was stained by immunofluorescence (indicated by the white triangle arrow in the figure). Refers to) It was observed that the expression of FN1 was significantly reduced as the concentration increased (as shown in Figure 3).

RPE cells were spread on fibronectin-pretreated slides, and different concentrations (0uM, 2uM, 5uM) of EGFR inhibitor erlotinib (MCE, HY-50896) were used to treat RPE cells for 48 hours, and extracellular fibers were observed. The zonulin component decreased with increasing erlotinib concentration (as shown in Figure 4).

RPE cells were treated with different concentrations (0uM, 2uM, 5uM) of EGFR inhibitor erlotinib (MCE, HY-50896) for 72 hours, and real-time quantitative PCR confirmed that erlotinib could significantly inhibit the expression of RPE FN1 and up-regulate the expression of BEST1 Expression (as shown in Figure 5), thus indicating that it can inhibit RPE fibrosis.

In vivo experiment:

Sodium hyaluronate gel (Shanghai Jianhua Fine Biological Products Co., Ltd., LOT: 22206281)/PBS mixture (50%, v/v) was injected into the subretinal cavity to cause long-term retinal detachment in the eyeballs of C57BL/6J mice, and It was confirmed that long-term retinal detachment would lead to discontinuity of the RPE layer (indicated by the white triangular arrow in the upper left of Figure 6), and fibrosis (increased expression of FN1, indicated by the white triangular arrow in the upper right of Figure 6). A small amount of microglial cells gathered in the subretinal space (white arrow in the lower left of Figure 6), and the expression of keratin increased (white arrow in the lower right of Figure 6). The animal model of the constructed subretinal membrane was confirmed to be consistent with the subretinal membrane cell types shown by human single-cell data.

Sodium hyaluronate gel (Shanghai Jianhua Fine Biological Products Co., Ltd., LOT: 22206281)/PBS mixture (50%, v/v) was injected into the subretinal cavity to cause long-term retinal detachment in the eyeballs of C57BL/6J mice, and It was confirmed that long-term retinal detachment would lead to fibrosis of RPE cells (increased expression of FN1, indicated by the white triangle arrow in the figure). On the 2nd day after modeling, the mice were gavaged with 40 mg/kg of erlotinib (MCE, HY-50896) or the carrier of the dissolved drug (DMSO), once a day for 7 consecutive days, and on the 10th day The eyeballs were taken the next day to compare the expression of FN1 in subretinal RPE cells (as shown in FIG. 7 ). It was found that erlotinib can significantly inhibit RPE fibrosis caused by retinal detachment (Fig. 7 bottom). The preventive effect of erlotinib on the subretinal membrane was confirmed.

Sodium hyaluronate gel (Shanghai Jianhua Fine Biological Products Co., Ltd., LOT: 22206281)/PBS mixture (50%, v/v) was injected into the subretinal cavity to cause long-term retinal detachment in the eyeballs of C57BL/6J mice, and It is confirmed that long-term retinal detachment will lead to fibrosis of RPE cells (increased expression of FN1, indicated by the white triangle arrow in the upper right of Figure 8), and migration and proliferation of retinal pigment epithelial cells (RPE65 thickens and presents multiple layers, as shown by the white triangle in the upper left of Figure 8 arrow pointing). On the 5th day after modeling, the mice were gavaged with 40 mg/kg of erlotinib (MCE, HY-50896) or the carrier of the dissolved drug (DMSO), and the mice were gavaged once a day for 7 consecutive days. Eyeballs were taken to compare the expressions of FN1 and RPE65 in subretinal RPE cells. It was found that erlotinib can significantly inhibit the RPE fibrosis caused by retinal detachment (lower right in Figure 8) and maintain the state of RPE65 monolayer (lower left in Figure 8). The therapeutic effect of erlotinib on the subretinal membrane was confirmed.

Therefore, it can be confirmed that erlotinib can prevent/or treat the generation of subretinal membrane after retinal detachment.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (1)

1. The application of erlotinib in the preparation of a medicament for preventing/treating subretinal membrane production after retinal detachment.

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