Application of erlotinib in preparation of integrated medicine for promoting retinal pigment epithelial cell transplantation

Abstract

The invention discloses an application of erlotinib in preparation of an integrated drug for promoting retinal pigment epithelial cell transplantation. It is found that human embryo RPE cells (human fRPE, hfRPE) transplanted to the subretinal space of a mouse specifically express EGFR, and after erlotinib intragastric administration (40mg / kg) treatment is performed on the mouse, it is observed that under the condition that an immunosuppressive agent is not used, xenogeneic donor cells can be better expanded in the subretinal space of the host and integrated into the RPE cells of the host, tight connection is formed, and therefore the immunosuppressive effect is achieved. Meanwhile, the RPE65 functional protein is stably expressed. Under the treatment of the erlotinib with the concentration, the integrity of tissue structures (retinal pigment epithelial cells, photoreceptor extracellular nodes and the like) of a subretinal space is not influenced.

Description

Technical Field

[0001] The invention belongs to a new field of medical applications, and particularly relates to the application of erlotinib in the preparation of an integration drug for promoting retinal pigment epithelial cell transplantation. Background Art

[0002] Retinal degenerative diseases are a spectrum of diseases with chronic dysfunction of RPE cells as the main pathological manifestation, including age-related macular degeneration (AMD), retinitis pigmentosa, Stargardt disease, etc., which are the main blinding diseases in people of all ages. Except for neovascular AMD, there is currently no effective treatment. Retinal pigment epithelial cell (RPE) transplantation is the most eye-catching research direction for the treatment of atrophic retinal degenerative diseases, and has shown gratifying therapeutic effects in many clinical trials around the world. However, in animals and long-term clinical observations, it was found that even under the long-term protection of immunosuppressants such as cyclosporine, the visual improvement brought about by RPE transplantation is not lasting, which is largely due to the chronic dysfunction and cell loss of the donor RPE. How to promote the functional integration and long-term survival of transplanted cells is an important challenge in RPE transplantation. Summary of the invention

[0003] The purpose of the present invention is to provide a new use of erlotinib for medicine, specifically to provide the use of erlotinib in the preparation of a drug for promoting integration after retinal pigment epithelial cell transplantation.

[0004] The present invention provides a new medical use of the existing drug erlotinib. Erlotinib is a reversible tyrosine kinase inhibitor that can inhibit the phosphorylation of intracellular tyrosine kinases related to a specific type of epidermal growth factor receptor (EGFR), and is a first-line drug for patients with locally advanced or metastatic non-small cell lung cancer with sensitive mutations in the EGFR gene. However, there has been no report on its use to promote the integration and function of transplanted RPE cells.

[0005] This study found that human fetal RPE cells (hfRPE) transplanted into the subretinal cavity of mice specifically expressed EGFR. After mice were treated with erlotinib (40 mg / kg) by oral gavage, it was observed that without the use of immunosuppressants, xenogeneic donor cells were better able to expand and integrate into the host's RPE cells in the subretinal cavity of the host, forming tight junctions and stably expressing RPE65 functional protein. Under this concentration of erlotinib treatment, the integrity of the subretinal cavity tissue structure (retinal pigment epithelial cells, photoreceptor cell outer segments, etc.) was not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 This is an immunofluorescence staining image of eyeball sections of normal mice and sodium iodate modeled mice in the examples.

[0007] Figure 2 This is an immunofluorescence staining image of eyeball sections of sodium iodate modeled mice injected with hfRPE cells in the subretinal space in the example.

[0008] Figure 3 This is an immunofluorescence staining image of eyeball sections of sodium iodate modeling mice treated with erlotinib in the example in which hfRPE cells were injected into the subretinal space.

[0009] Figure 4 This is an immunofluorescence staining image of the eyeball RPE / choroid flat mount of the sodium iodate modeling mice treated with erlotinib in the example in which hfRPE cells were injected into the subretinal space.

[0010] Figure 5 This is an immunofluorescence staining image of eyeball sections of sodium iodate modeling mice treated with erlotinib in the example in which hfRPE cells were injected into the subretinal space.

[0011] Figure 6 This is an immunofluorescence staining image of the eyeball RPE / choroid flat mount of the sodium iodate modeling mice treated with erlotinib in the example in which fRPE cells were injected into the subretinal space. DETAILED DESCRIPTION

[0012] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0013] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0014] In vitro experiments:

[0015] The hfRPE cells used for transplantation were transfected with lentivirus (Gene Gene, GCNL0397792) to express mCherry fluorescent protein, thereby specifically distinguishing donor cells from host cells.

[0016] In vivo experiments:

[0017] Sodium iodate solution was injected into the tail vein of mice at a concentration of 15 mg / kg to construct a mouse model of retinal degeneration with RPE cell-photoreceptor cell damage. After the modeling, the eyeballs of the mice were collected and the thinning of the retinal outer nuclear layer, the decrease in the number of RPE cells and the decrease in the expression of RPE65 were observed.

[0018] By injecting hfRPE cells expressing mCherry fluorescent protein into the subretinal space of sodium iodate mice to simulate retinal degenerative disease cell replacement therapy, the ocular tissue sections were stained on the third day after transplantation to confirm the specific increase in the expression of EGFR in the transplanted cells. This confirmed that the EGFR signaling pathway of donor cells was specifically upregulated in the transplanted disease microenvironment.

[0019] hfRPE cells expressing mCherry fluorescent protein were injected into the subretinal cavity of sodium iodate mice to simulate cell replacement therapy for retinal degenerative diseases. On the second day after modeling, mice were gavaged with 40 mg / kg of EGFR inhibitor erlotinib (MCE, HY-50896) or a carrier (DMSO) that dissolved the drug, once a day for 7 consecutive days. On the 7th day of gavage, the eyeballs were taken for section immunofluorescence staining, and it was found that compared with the control group, which formed obvious cell clumps, erlotinib could promote better dispersion of donor cells in the subretinal cavity area, thereby facilitating their integration into host RPE cells and promoting their expression of RPE65 functional protein.

[0020] hfRPE cells expressing mCherry fluorescent protein were injected into the subretinal cavity of sodium iodate mice to simulate cell replacement therapy for retinal degenerative diseases. On the second day after modeling, mice were gavaged with 40 mg / kg of EGFR inhibitor erlotinib (MCE, HY-50896) or a carrier (DMSO) that dissolved the drug, once a day for 7 consecutive days. On the 7th day of gavage, the eyeballs were taken for immunofluorescence staining of RPE-choroid flat sheets, and it was found that compared with the control group, which formed obvious cell clumps, erlotinib could promote the integration of donor cells into host RPE cells, and at the same time form ZO-1+ tight connections with host cells to maintain the hexagonal structure.

[0021] hfRPE cells expressing mCherry fluorescent protein were injected into the subretinal cavity of sodium iodate mice to simulate cell replacement therapy for retinal degenerative diseases. On the second day after modeling, mice were gavaged with 40 mg / kg of EGFR inhibitor erlotinib (MCE, HY-50896) or a vehicle (DMSO) that dissolved the drug, once a day for 7 consecutive days. On the 7th day, the eyeballs were taken for section immunofluorescence staining, and it was found that compared with the control group, the transplanted cells had obvious fibrosis (increased FN1 expression), and erlotinib could significantly inhibit the fibrosis of donor cells.

[0022] By injecting fRPE cells expressing mCherry fluorescent protein into the subretinal cavity of sodium iodate mice to simulate cell replacement therapy for retinal degenerative diseases, mice were gavaged with 40 mg / kg of EGFR inhibitor erlotinib (MCE, HY-50896) or a carrier (DMSO) for dissolving the drug starting on the second day after modeling, once a day for 7 consecutive days. On the 7th day of gavage, the eyeballs were taken for immunofluorescence staining of RPE-choroid flat sheets, and it was found that compared with the control group, the expression of RPE65 in donor cells was weak, with obvious boundaries from surrounding cells and a significant decrease in number. Erlotinib can promote the survival of donor cells and high expression of RPE65 functional protein. It is confirmed that erlotinib is beneficial to maintain the number of donor RPE cells and perform normal functions.

Claims

1. Application of erlotinib in the preparation of drugs for promoting integration after retinal pigment epithelial cell transplantation.

Images