Application of lomitapil

Abstract

The invention discloses application of lomitapil, and relates to the field of medicines. The invention relates to an application of lomitapil as an SRSF7 protein inhibitor drug. The lomitapil is used for preparing the medicine for treating the pulmonary fibrosis disease. The invention provides the cardiac application of lomitapil in treatment of pulmonary fibrosis diseases, and the lomitapil has the advantages of low research cost, low risk, high success rate and high clinical test speed.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of lomitapipim as a SRSF7 protein inhibitor drug. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with irreversible damage to lung tissue structure and function, high mortality and poor prognosis. IPF is characterized by inflammatory damage and destruction of tissue structure, which causes abnormal repair and ultimately leads to terminal changes in the lungs. The severity of IPF is comparable to that of lung cancer, seriously affecting the quality of life of patients and significantly increasing the family and socioeconomic burden. IPF is the result of multiple interactions of genetic and environmental risk factors. In addition to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and sarcoidosis that can cause pulmonary fibrosis, drugs, environment, infection, and genetics can also cause pulmonary fibrosis. The awareness rate of this disease is relatively low, resulting in many patients in remote areas not being diagnosed and treated in a timely manner.

[0003] Therefore, finding more effective treatments is of great significance to improving patients' quality of life and reducing their economic burden. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the present invention provides the use of lomitapide as a SRSF7 protein inhibitor drug.

[0005] Furthermore, the lomitapir is used to prepare a medicine for treating pulmonary fibrosis.

[0006] Compared with the prior art, the present invention has the following technical effects: 1. Lomitapipri is a drug that inhibits SRSF7 and is a new lipid-lowering drug for the treatment of homozygous familial hypercholesterolemia (HoFH). The present invention applies lomitapri to drugs for the treatment of pulmonary fibrosis, expanding the indications of drugs already on the market. Compared with the development of new drugs, the reuse of lomitapri has the obvious advantages of low research cost, low risk, high success rate, and fast clinical trial speed, and can be used as a potential drug or combination drug for the treatment of IPF.

[0007] 2. Experiments on mice have shown that the present invention can reduce collagen deposition in lung tissue of mice with pulmonary fibrosis, improve the respiratory function of mice with pulmonary fibrosis, restore the lung structure of mice, and inhibit the transformation of lung fibroblasts into myofibroblasts, thus having significant technical effects.

[0008] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 In a specific embodiment of the present invention, an in vitro myocardial fibrosis model is constructed, wherein: Figure 1 A is the result of qRT-PCR showing the high expression level of SRSF7 mRNA in the mouse fibrosis model induced by Bleomycin (BLM); Figure 1 B is the result of immunohistochemical staining showing that the expression of SRSF7 is significantly increased in patients with pulmonary fibrosis; Figure 1 C is the result of Western Blot showing the high expression of SRSF7 protein in the lung tissue of IPF patients; Figure 2 In a specific embodiment of the present invention, the results of the detection of the effects of overexpression of SRSF7 on the respiratory function and pulmonary fibrosis in mice are shown. Figure 2 A is the mRNA expression of SRSF7 in mice treated with AAV-NC and AAV-SRSF7, n=5, *P<0.05, Figure 2 B: IC and FVC were detected in mice treated with AAV-NC and AAV-SRSF7, n=5, **P<0.01, Figure 2 C shows the fibrosis of mouse lungs observed using micro-CT, n=5; Figure 3 In a specific embodiment of the present invention, the expression results of SRSF7 overexpression on fibrosis-related proteins and genes in mice are shown. Figure 3 A is the mRNA expression of liver fibrosis-related genes in mice, n=5, *P<0.05, **P<0.01, Figure 3 B is the expression of FN1, α-SMA and SRSF7 proteins in mice, n=5, *P<0.05, **P<0.01; Figure 4 In one specific embodiment of the present invention, the results of the effect of overexpression of SRSF7 on lung inflammation and fibrosis in mice are shown. Figure 4 A: H&E and Masson staining were used to detect the collagen content in lung tissue, n=5, scale bar=50μm, Figure 4 B is the protein expression of collagen 1α1 and α-SMA in mice, n=5, scale bar=50μm; Figure 5 In a specific embodiment of the present invention, the overexpression of SRSF7 promotes the expression of fibrosis-related proteins and genes. Figure 5 A is the result of qRT-PCR experiment, n=6, **P<0.01. Figure 5 B is the expression results of fibrosis-related gene mRNA, n=6, **P<0.01, *P<0.05, Figure 5 C is the Western blot result, n=4, *P<0.05; Figure 6 In a specific embodiment of the present invention, overexpression of SRSF7 affects the proliferation and migration of human embryonic lung fibroblasts. Figure 6 A is EdU experiment (scale bar=50μm, n=4, **P<0.01), Figure 6 B is the wound healing experiment (scalebar=200μm, n=4, *P<0.05); Figure 7 In a specific embodiment of the present invention, the results of the effect of overexpression of SRSF7 on transdifferentiation of human embryonic lung fibroblasts; Figure 8 It is the result of the effect of overexpression of SRSF7 on the contractility of fibroblasts in a specific embodiment of the present invention; Fig. 9 In a specific embodiment of the present invention, the present invention is a flow chart of a test scheme for administering the SRSF7 inhibitor lomitapid and the control drug pirfenidone to animals, wherein: Fig. 9 A is a specific operation flow chart; Fig. 9 B is the Western Blot result. Fig. 9 C is the result of qRT-PCR experiment; Fig.10 In a specific embodiment of the present invention, the results of administering lomitapir and the control drug pirfenidone to animals, wherein: Fig.10 A is the result of lung CT examination. Fig.10 BC is the result of respiratory test on mice; Fig.11 In a specific embodiment of the present invention, the pathological section results of animals treated with lomitapide and the control drug pirfenidone are shown in FIG. Fig.11 A is the result of Masson staining. Fig.11 B is the result of H&E staining. DETAILED DESCRIPTION

[0010] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0011] 1. Establishment of mouse fibrosis model The experimental mice used C57BL / 6 male mice (6-8 weeks old, about 20g) were purchased from Liaoning Changsheng Biological Experimental Animal Technology Co., Ltd.; the breeding conditions of C57BL / 6 mice were as follows: the temperature was set at 25°C, the humidity was 45%RH, the light / darkness was alternating for 12 hours, and the ventilation was 24 hours.

[0012] Mice weighing about 25g and aged 6-8 weeks were selected and anesthetized by intraperitoneal injection of aflototin (250mg / kg). After disinfection, the skin of the mouse throat was cut open and bleomycin (BLM 3mg / kg) or an equal amount of saline (Saline), 3 mg / kg body weight, was injected into the trachea to construct an animal model. After 21 days, a mouse pulmonary fibrosis model was formed. The mice injected with bleomycin were designated as the BLM group, and the mice injected with saline were designated as the Sham group.

[0013] 2. Expression detection of splicing factor SRSF7 in pulmonary fibrosis tissue SRSF7 (also known as 9G8) is a member of the serine-arginine family of splicing factors. Its signaling pathway not only plays a key role in tumor occurrence, progression, and metastasis, but also mediates age-related alternative splicing events, making SRSF7 a highly potential therapeutic target for age-related chronic diseases.

[0014] The qRT-PCR results of mice in the fibrosis model (n=6) are as follows Figure 1 As shown in A, the qRT-PCR results showed that compared with the Sham group, the expression of fibrosis-related marker FN1 in the lung tissue of BLM-treated mice was increased, and the expression of splicing factor SRSF7 was significantly upregulated.

[0015] Pathological sections of lung tissues from IPF patients and paraffin sections of lung tissues from healthy controls were obtained for immunohistochemical staining of SRSF7. Figure 1 As shown in B, the expression of SRSF7 in the IPF group was significantly upregulated compared with the non-IPF group. Proteins extracted from lung tissues of IPF patients and healthy controls were subjected to western blot detection. The test results are shown in Figure 1 As shown in C, the results also showed that SRSF7 expression was significantly upregulated in IPF patients.

[0016] It can be seen that the splicing factor SRSF7 is highly expressed in pulmonary fibrosis tissue, so SRSF7 is a risk factor for IPF.

[0017] 3. Verification of SRSF7 promoting pulmonary fibrosis in mice 1). Samples were collected on the 21st day after intratracheal instillation of adeno-associated virus-5 (AAV5-SRSF7) into mice for qRT-PCR experiments. The results are as follows: Figure 2 As shown in A, after the overexpression efficiency of SRSF7 was verified by qRT-PCR, the respiratory function of mice was tested using a small animal lung function tester. Figure 2 As shown in Figure B, the results showed that AAV-SRSF7 treatment can significantly reduce the forced vital capacity (FVC) and inspiratory capacity (IC) of mice, indicating that SRSF7 alone can lead to respiratory dysfunction in mice. The effect of SRSF7 on pulmonary fibrosis in mice was observed using small animal CT. Figure 2 As shown in C, partial fibrotic areas can be observed in the lungs of mice after administration of SRSF7, indicating that the mice had pulmonary fibrosis lesions, indicating that overexpression of SRSF7 induced respiratory function decline and pulmonary fibrosis in mice.

[0018] 2). Detection of fibrosis-related markers in mice intratracheally instilled with AAV-SRSF7. The qRT-PCR results are as follows: Figure 3 As shown in A, the mRNA levels of FN1, COL1α1, COL3α1 and ACTA2 were significantly increased in the lung tissue of mice injected with AAV-SRSF7 intratracheally. Figure 3 As shown in B, the expression of FN1 and α-SMA proteins in lung tissue was upregulated after intratracheal instillation of AAV-SRSF7. The above results indicate that overexpression of SRSF7 significantly promotes the expression of fibrosis markers in lung tissue.

[0019] 3). Pathological staining of mouse lung tissue sections, Masson and H&E staining results are as follows Figure 4 A and Figure 4 As shown in B, intratracheal instillation of AAV-SRSF7 led to the destruction of mouse lung tissue structure, collagen fiber deposition, and inflammatory infiltration. The results of COL1α1 and α-SMA immunohistochemical staining showed that intratracheal instillation of AAV-SRSF7 could increase collagen deposition in mouse lung tissue, and α-SMA expression was significantly upregulated, suggesting that SRSF7 can promote the transdifferentiation of mouse lung fibroblasts into myofibroblasts.

[0020] 4). Construct SRSF7 overexpression plasmid to overexpress SRSF7 in human embryonic lung fibroblasts MRC-5, such as Figure 5 As shown in A, the overexpression efficiency of SRSF7 in fibroblasts was verified. The qRT-PCR results are shown in Figure 5As shown in B, overexpression of SRSF7 in MRC-5 significantly increased the mRNA levels of fibrosis-related genes FN1, COL1α1, COL3α1, and ACTA2; Western blot experimental results are shown Figure 5 As shown in Figure C, compared with the control group, overexpression of SRSF7 can promote the expression of FN1 and α-SMA proteins in MRC-5 cells. The above results show that overexpression of SRSF7 can induce excessive activation and transdifferentiation of fibroblasts, leading to fibrosis of cells.

[0021] 5). Through EdU cell proliferation experiment, it can be seen that overexpression of SRSF7 can significantly promote fibroblast proliferation. Figure 6 A. At the same time, the results of the cell scratch experiment are shown in Figure 6 As shown in Figure B, the effect of SRSF7 on the migration ability of fibroblasts was verified. The results showed that overexpression of SRSF7 could significantly increase the migration ability of cells.

[0022] 6). During chronic injury or persistent inflammation, fibroblasts are abnormally activated and differentiate into myofibroblasts. Their abnormal activation can lead to disordered extracellular matrix production, promote local scar formation, and ultimately lead to respiratory failure and loss of gas exchange function in the patient's lungs. In some cases, it also contributes to tumorigenesis. The main characteristics of myofibroblasts are contractility, expression of α-SMA, and excessive production of extracellular matrix proteins. Immunofluorescence experiments were performed, and the results were as follows: Figure 7 As shown in the results, overexpression of SRSF7 can promote its nuclear accumulation and significantly upregulate the expression of α-SMA protein, thereby promoting the transdifferentiation of fibroblasts into myofibroblasts.

[0023] 7). Since the differentiation of fibroblasts into myofibroblasts is accompanied by the enhancement of cell contraction characteristics, we further verified the effect of SRSF7 on the contraction ability of fibroblasts through collagen gel contraction experiments. The results showed that SRSF7 can significantly promote the contraction of fibroblast collagen. Figure 8 shown.

[0024] 4. Lomitapipim can inhibit the expression of SRSF7 and the expression of pulmonary fibrosis-related markers Lomitapide is a new lipid-lowering drug for the treatment of homozygous familial hypercholesterolemia (HoFH). It is approved by the U.S. Food and Drug Administration (FDA) for the treatment of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), apolipoprotein B (apoB) and non-high-density lipoprotein cholesterol (Non-HDL-C) levels in HoFH patients. Studies have shown that lomitapide can protect focal cerebral ischemic nerves by increasing autophagic flux and inhibiting apoptosis, repositioning lomitapide as a potential therapeutic drug for the treatment of ischemic stroke. In this embodiment, lomitapide is a purchased drug, and the manufacturer and product number are: Lomitapide Mesylate, Selleck, S7633.

[0025] On the seventh day after the experimental mice were injected with bleomycin, they were given lomitapipir or pirfenidone by gavage, and the mouse lung tissues were isolated on the 21st day.

[0026] Western blot experiments were performed on the mouse tissues. The experimental process is as follows: Fig. 9 As shown in A, the Western blot results are as follows Fig. 9 As shown in B, β-actin is the internal reference. It can be seen that lomitapid inhibits the expression of SRSF7 and can also inhibit the expression of mouse fibrosis markers FN1 and α-SMA. The qRT-PCR experiment was performed on the resistance of mice. The results of the qRT-PCR experiment are shown in Fig. 9 As shown in C, lomitapipride can inhibit the mRNA expression of fibrosis-related marker genes Fn1, Collagen 1α1 and ACTA2 and SRSF7, reduce the levels of SRSF7 protein and mRNA in lung fibroblasts, and inhibit the transformation of lung fibroblasts into myofibroblasts, providing new intervention means and theoretical basis for improving the treatment of model-induced and damaged lung fibroblasts.

[0027] 5. Lomitapide partially restored bleomycin-induced respiratory function impairment in mice The mice given lomitapipim and pirfenidone underwent lung CT examination. The results were as follows Fig.10 As shown in A, the test results showed that lomitapide can reduce BLM-induced lung fibrosis lesions and reduce the area of ​​ground-glass fibrosis in the lung induced by BLM. Lomitapide can significantly reduce the increase in the shadow area of ​​mouse lung tissue caused by bleomycin, thereby significantly reducing the shadow area of ​​the mouse lung.

[0028] The mice were tested for respiratory function after administration. Fig.10As shown in BC, the results showed that lomitapide could partially restore the respiratory function of mice inhibited by bleomycin, alleviate the respiratory function impairment of mice induced by bleomycin, increase the forced vital capacity and deep inspiratory volume of mice, and significantly improve the respiratory function impairment and pulmonary fibrosis induced by bleomycin in mice.

[0029] 6. Lomitapipimide reduces bleomycin-induced lung tissue infiltration and scar formation in mice After the mice were euthanized, the lung tissues were taken out for pathological sectioning and Masson staining. The results of Masson staining are shown in Fig.11 As shown in A, lomitapipride can limit the reduction of collagen deposition in mouse lung tissue.

[0030] H&E staining was performed on the pathological sections of mouse lung tissue. Fig.11 As shown in B, the staining results showed that lomitapide could significantly reduce the inflammatory infiltration in the lung tissue of mice.

[0031] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Application of lomitapide as a SRSF7 protein inhibitor drug.

2. The use according to claim 1, characterized in that The lomitapir is used for preparing medicine for treating pulmonary fibrosis.

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