CN115414486A

CN115414486A - VEGFA signal and FGF-2 signal double-effect inhibitor and new application of FGF-2 signal inhibitor

Info

Publication number: CN115414486A
Application number: CN202210945835.9A
Authority: CN
Inventors: 杨云龙; 陈瑞波; 叶颖
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2022-08-08
Filing date: 2022-08-08
Publication date: 2022-12-02

Abstract

The application provides a VEGFA signal, an FGF-2 signal double-effect inhibitor and new application of the FGF-2 signal inhibitor. Use of a VEGFA signalling and a dual effect inhibitor of FGF-2 signalling and/or an inhibitor of FGF-2 signalling for the preparation of a medicament for the treatment of tumours which are resistant to inhibitors of VEGFA signalling. The medicine can inhibit drug resistance of tumor to VEGFA signal inhibitor, and improve therapeutic effect on tumor.

Description

VEGFA signal and FGF-2 signal double-effect inhibitor and new application of FGF-2 signal inhibitor

Technical Field

The application relates to the technical field of medicines, in particular to a VEGFA signal, an FGF-2 signal double-effect inhibitor and new application of the FGF-2 signal inhibitor.

Background

Nasopharyngeal carcinoma is a malignant epithelial tumor which can be classified into keratinizing squamous cell carcinoma, non-keratinizing squamous cell carcinoma, and undifferentiated or poorly differentiated carcinoma. Because nasopharyngeal carcinoma occurs in a special anatomical location, radiotherapy is the main means for treating non-metastatic nasopharyngeal carcinoma at present. In recent years, the combination of adjuvant chemotherapy and radiotherapy can significantly prolong the overall survival rate of patients. For other malignant solid tumors, anti-angiogenesis drugs such as VEGFA signal inhibitor and the like are usually adopted clinically to treat patients, and the survival time of the patients can be effectively improved. However, clinical trials have shown that inhibitors of VEGFA signaling such as bevacizumab are not effective in the treatment of nasopharyngeal cancer, probably due to their resistance to VEGFA signaling inhibitors.

Therefore, there is a need to provide a method for treating nasopharyngeal carcinoma that is resistant to VEGFA signaling inhibitors, and to provide a method for improving the resistance of nasopharyngeal carcinoma to VEGFA signaling inhibitors, thereby increasing the dosage options for patients with nasopharyngeal carcinoma and improving the survival of the patients.

Tumor angiogenesis is important for the growth and metastasis of tumors. The main signaling molecules mediating angiogenesis include VEGFA, PDGF, FGF and the like. They act on the corresponding receptors of endothelial cells in tumors, activating downstream signaling pathways, and promoting angiogenesis. The VEGFA signal inhibitor can inhibit angiogenesis by blocking VEGFA ligand and/or receptor and/or downstream signal, effectively block the supply of oxygen and nutrient substances in tumor, and achieve the purpose of treating tumor. In some cases, however, tumors compensate for maintaining angiogenesis by expressing other types of angiogenic signals, resulting in resistance to inhibitors of VEGFA signaling.

Disclosure of Invention

In view of the above, the present application provides an application of VEGFA signaling and FGF-2 signaling dual-effect inhibitor and/or FGF-2 signaling inhibitor in the preparation of a medicament for treating tumor, and an application of FGF-2 signaling inhibitor in the preparation of a medicament for improving the drug resistance of tumor to VEGFA signaling inhibitor.

In a first aspect, the present application provides the use of VEGFA signaling and a dual-effect inhibitor of FGF-2 signaling and/or an inhibitor of FGF-2 signaling for the preparation of a medicament for the treatment of tumors that are resistant to inhibitors of VEGFA signaling.

Optionally, the dual-effect inhibitor for VEGFA signaling and FGF-2 signaling can inhibit VEGFA signaling pathway and FGF signaling pathway of endothelial cells in tumors at the same time, thereby overcoming drug resistance of the tumors to the VEGFA signaling inhibitor and inhibiting tumor angiogenesis.

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises rivastigmine.

Optionally, the FGF-2 signaling inhibitor inhibits at least one of an FGF-2 ligand, an FGFR1 receptor, and a downstream signaling pathway key molecule MYC thereof, and the FGF-2 signaling inhibitor comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug.

Optionally, the VEGFA signaling inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and downstream signaling pathway key molecule MYC thereof, and the VEGFA signaling inhibitor comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs.

Optionally, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling as a single active ingredient or with other pharmaceutically acceptable active ingredients constitutes the medicament.

Alternatively, the FGF-2 signalling inhibitor as a single active ingredient or together with other pharmaceutically acceptable active ingredients constitutes the medicament.

Optionally, the tumor comprises a solid tumor comprising at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, clear cell renal cancer, skin melanoma, fibroids, and nasopharyngeal cancer.

Further, the solid tumor comprises nasopharyngeal carcinoma.

Optionally, the tumor is a solid tumor expressing FGF-2.

In a second aspect, the present application provides the use of an FGF-2 signalling inhibitor for the preparation of a medicament for improving the resistance of a tumour to a VEGFA signalling inhibitor, said tumour being resistant to a VEGFA signalling inhibitor.

Further, the solid tumor comprises nasopharyngeal carcinoma.

Alternatively, the tumor is a solid tumor expressing FGF-2.

In a third aspect, the present application provides a medicament for treating a tumor that is resistant to a VEGFA signaling inhibitor, the medicament comprising VEGFA signaling and at least one of a dual-effect inhibitor of FGF-2 signaling and an inhibitor of FGF-2 signaling.

In a fourth aspect, the present application provides a medicament for improving the resistance of a tumour to a VEGFA signalling inhibitor, said medicament comprising an FGF-2 signalling inhibitor.

Advantages of embodiments of the present application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the present application.

Drawings

FIG. 1 is a graph showing the comparison of the expression levels of pro-angiogenic factors in various tumors, wherein A is a graph showing the comparison of the expression levels of pro-angiogenic factors in tumor RNA expression profiles, and B is a graph showing the comparison of the relative expression levels of FGF-2 in tumor cell lines.

FIG. 2 shows the experimental results of drug resistance of nasopharyngeal carcinoma tumor-bearing mice to VEGFA signal inhibitor, wherein A is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells, B is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells knocked down with FGF-2 gene, C is the tumor vascular density result, and D is the tumor lung metastasis result.

FIG. 3 is a graph of the results of the effect of FGF-2 in fibroids and breast cancer, where A is the tumor volume of the fibroid tumor-bearing mouse, B is the tumor vasculature density results of the fibroid tumor-bearing mouse, C is the tumor lung metastasis results in the fibroid tumor-bearing mouse, D is the tumor volume of the breast cancer tumor-bearing mouse, E is the tumor vasculature density results in the breast cancer tumor-bearing mouse, and F is the tumor lung metastasis results in the breast cancer tumor-bearing mouse.

FIG. 4 is a graph of the effect of a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling, where A is tumor volume, B is the tumor vascular density results, and C is the tumor lung metastasis results.

Detailed Description

While the following is a preferred embodiment of the embodiments of the present application, it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the embodiments of the present application, and such improvements and modifications are also considered to be within the scope of the embodiments of the present application.

The application provides an application of VEGFA signals and FGF-2 signal double-effect inhibitors and/or FGF-2 signal inhibitors in preparing drugs for treating tumors, wherein the tumors have drug resistance to the VEGFA signal inhibitors. That is, in one embodiment, the medicament may include a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling; in another embodiment, the medicament may comprise an inhibitor of FGF-2 signaling; in yet another embodiment, the medicament may include a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling, and an inhibitor of FGF-2 signaling.

In the embodiments of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling can simultaneously inhibit VEGFA signaling pathway and FGF signaling pathway of endothelial cells in tumors, thereby overcoming drug resistance of tumors to VEGFA signaling inhibitors and inhibiting tumor angiogenesis. The VEGFA signal and FGF-2 signal double-effect inhibitor inhibits expression of VEGFA signals, FGF-2 signals and downstream signal molecules such as MYC, so that proliferation of vascular endothelial cells is inhibited, angiogenesis is inhibited, proliferation of tumor cells is inhibited, tumor volume is reduced, apoptosis of the tumor cells is promoted, the effect of treating tumors is achieved, tumors resistant to the VEGFA signal inhibitor can be effectively treated, and the treatment effect of the existing tumors is optimized.

In the present application, FGF-2 signaling inhibitors are effective in improving, treating (eliminating) tumor resistance to VEGFA signaling inhibitors. Specifically, the FGF-2 signal inhibitor inhibits the expression of downstream signal molecules such as MYC through a receptor of FGF-2 on tumor endothelial cells, so that the proliferation of the vascular endothelial cells is inhibited, the angiogenesis is hindered, the proliferation of the tumor cells is inhibited, the tumor volume is reduced, the apoptosis of the tumor cells is promoted, and the effect of treating tumors is achieved.

In embodiments of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug that simultaneously inhibits both VEGFA signaling and FGF-2 signaling pathways. In one embodiment, the chemical agent of the dual effect inhibitor may be a small molecule organic compound. In another embodiment, the proteinaceous agents of the dual effect inhibitor include, but are not limited to, native proteins, recombinant proteins, and the like. In yet another embodiment, the gene-based agents of the dual effect inhibitor include, but are not limited to, nucleic acid fragments, such as DNA fragments, RNA fragments. For example, the gene-based drug of the dual-effect inhibitor can be an siRNA drug for silencing the expression of ligands and/or receptors and/or downstream signaling molecules in the VEGFA signaling pathway and the FGF-2 signaling pathway, and can also be an shRNA plasmid or shRNA lentiviral particle for down-regulating the protein expression of a target gene.

In the examples herein, the VEGFA signaling and FGF-2 signaling dual-effect inhibitor comprises lenvatinib.

In an embodiment of the application, the FGF-2 signaling inhibitor inhibits at least one of an FGF-2 ligand, an FGFR1 receptor, and a downstream signaling pathway key molecule MYC thereof, and the FGF-2 signaling inhibitor comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug.

In one embodiment, the chemical agent of the FGF-2 signaling inhibitor may be a small molecule organic compound. For example, the chemical agents of FGF-2 signaling inhibitors include FGFR1 tyrosine kinase inhibitor AP 245634, which inhibits FGF-2 signaling, and the like. In another embodiment, the proteinaceous agents of the FGF-2 signaling inhibitor include, but are not limited to, native proteins, recombinant proteins, and the like. Protein drugs such as FGF-2 signaling inhibitors include polypeptides that block FGF-2 binding to FGFR such as FR-1039 and the like. In yet another embodiment, the gene-based agents of the FGF-2 signaling inhibitors include, but are not limited to, nucleic acid fragments, e.g., DNA fragments, RNA fragments. For example, the gene-based agent of the FGF-2 signaling inhibitor can be an siRNA agent that silences expression of a ligand and/or receptor and/or downstream signaling molecule in the FGF-2 signaling pathway, and can also be an shRNA plasmid or shRNA lentiviral particle that down-regulates protein expression of a target gene.

In embodiments of the present application, the VEGFA signaling inhibitor inhibits at least one of a VEGFA ligand, a VEGFR2 receptor, and a downstream signaling pathway key molecule MYC thereof, and the VEGFA signaling inhibitor includes at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug.

In an embodiment of the present application, the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is used as a single active ingredient or is combined with other pharmaceutically acceptable active ingredients to form a medicament.

In an embodiment of the present application, the FGF-2 signaling inhibitor is present as a single active ingredient or is present as a pharmaceutical with other pharmaceutically acceptable active ingredients. In one embodiment, the other pharmaceutically acceptable active ingredient in the medicament comprises an inhibitor of VEGFA signaling.

In embodiments of the present application, the tumor comprises a solid tumor. In an embodiment of the present application, the solid tumor includes at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroma, and nasopharyngeal carcinoma. In one embodiment, the solid tumor comprises nasopharyngeal carcinoma. The VEGFA signaling and FGF-2 signaling dual-effect inhibitor can effectively treat nasopharyngeal carcinoma resistant to the VEGFA signaling inhibitor, and the FGF-2 signaling inhibitor can treat the nasopharyngeal carcinoma resistant to the VEGFA signaling inhibitor. Nasopharyngeal carcinoma is characterized in that tumor cells thereof highly express FGF-2; FGF-2 can activate the expression of downstream signal molecules such as MYC and the like through one of FGFR1 highly expressed in endothelial cells, so that the endothelial cell proliferation and angiogenesis are alternatively activated, and the drug resistance effect of the VEGFA signal inhibitor on angiogenesis cannot be inhibited.

In the present examples, the tumor is a solid tumor expressing FGF-2. Furthermore, the FGF-2 is highly expressed by the solid tumor, and the targeting property of the medicament is improved. In the present examples, the tumor is a solid tumor expressing VEGFA. In the examples of the present application, the tumor is a solid tumor expressing FGF-2 and VEGFA.

In the present embodiment, the medicament may further include a pharmaceutically acceptable carrier and/or adjuvant. The medicament simultaneously contains VEGFA signals, FGF-2 signal double-effect inhibitors and/or FGF-2 signal inhibitors, and pharmaceutically acceptable carriers and/or auxiliary materials. In the present application, the "pharmaceutically acceptable carrier" is used to transport the drug in the present application to exert its intended effect. In general, delivery is from one organ or portion to another, and the carrier must be compatible with the pharmaceutical composition, not interfere with the biological activity of the drug, and be relatively non-toxic, e.g., the carrier enters the body without causing toxic side effects or having a severe reaction with the drug it carries, which does not adversely affect the patient.

In the present embodiment, the pharmaceutically acceptable carrier includes at least one of a solvent, a polymer, a liposome, a recombinant viral vector and a eukaryotic recombinant expression vector, but is not limited thereto. In one embodiment, the solvent includes, but is not limited to, at least one of water, physiological saline, and other non-aqueous solvents. In another embodiment, the polymer includes one or more of polylysine, polyethyleneimine (branched and/or linear) and its modifications, polyamidoamine dendrimer (PAMAM) and its derivatives, polypropyleneimine dendrimer (PPI) and its derivatives, chitosan, polylactic-co-glycolic acid (PLGA), polylactic acid, gelatin, cyclodextrin, sodium alginate, albumin, and hemoglobin, but is not limited thereto. Among them, polyethyleneimine and its modified products, PAMAM and its derivatives, PPI and its derivatives, chitosan, etc. may be referred to as cationic polymers. In another embodiment, the liposome can be self-assembled from cationic lipid, neutral auxiliary lipid, cholesterol, phospholipid (such as soybean lecithin, egg yolk lecithin, cephalin, etc.), or formed by inserting distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) into phospholipid layer formed by phospholipid molecules. In another embodiment, the recombinant viral vector may include one or more of a lentiviral vector, an adenoviral vector, and a retroviral vector, but is not limited thereto.

In the present application, the VEGFA signal and the FGF-2 dual-effect inhibitor and/or FGF-2 inhibitor in the drug may be dispersed or adsorbed in the above-mentioned carrier to form a dispersion system, or may be coated/encapsulated by the above-mentioned liposome, polymer, etc. to form a spherical structure (e.g., nanocapsule or microcapsule). For example, while the VEGFA signaling and dual-effect inhibitor of FGF-2 signaling and/or inhibitor of FGF-2 signaling can be nucleic acid fragments, drugs can include, but are not limited to, cationic polymers, polypeptides, protein drugs, and the like, that encapsulate, bind to, or blend with the nucleic acid fragments. The active ingredients encapsulated in the spherical structure can be subjected to slow release, controlled release or targeted release, so that the medicament can exert the optimal efficiency and the stability of the medicament can be improved. For example, albumin, gelatin, chitosan, polylactic acid may generally form microspheres that can disperse or encapsulate a pharmaceutically active ingredient.

In an embodiment of the present application, the excipient comprises at least one of a diluent and an excipient. The primary function of the diluent is to fill the weight or volume of the tablet to facilitate tableting. In one embodiment, the diluent comprises one or more of starches, sugars, celluloses, and inorganic salts. Excipients refer to additives in a drug in addition to the primary pharmaceutically active ingredient. In one embodiment, the excipients include, for example, binders in tablets, fillers, disintegrants, lubricants, wines in pills, vinegar, juices, etc., base parts in semi-solid preparations ointments, creams, preservatives in liquid preparations, antioxidants, flavors, fragrances, solubilizers, emulsifiers, solubilizers, tonicity adjusting agents, colorants, etc.

In the present embodiment, the form of the drug includes tablets, capsules, powders, granules, pills, syrups, solutions or suspensions. The specific application form depends on the actual situation. In the present embodiment, the drug may be administered orally or by injection. For administration by injection, the pharmaceutical form is preferably a solution, for example dissolved in water or physiological saline. In one embodiment, the injection may be administered intraperitoneally, subcutaneously, intramuscularly, or intravenously. In the present embodiment, the drug may be administered locally or systemically.

In the present embodiment, the amount of the drug is 0.1 to 100 mg/kg body weight per day. In particular, the amount of drug employed will depend upon a variety of factors including, but not limited to, the desired biological activity and tolerance of the subject to the drug.

Nasopharyngeal carcinoma naturally has high expression of FGF-2, and in nasopharyngeal carcinoma or FGF-2 high expression solid tumors, a series of molecular biological experiments prove that FGF-2 signals can act on FGFR1 receptors on endothelial cells in tumors, activate downstream signal pathways such as MYC and the like, and promote vascular endothelial cell proliferation. The classical VEGFA signal can act on VEGFR2 receptors on endothelial cells in tumors, activate downstream signal pathways such as MYC and the like, and promote the proliferation of vascular endothelial cells. Since both signaling pathways share the same downstream signaling molecule, FGF-2 can compensatory maintain angiogenesis in the presence of suppressed VEGFA signaling, resulting in tumor resistance to VEGFA signaling inhibitors. The VEGFA signal and FGF-2 signal double-effect inhibitor can inhibit the two signals simultaneously, and breaks the drug resistance mechanism of tumors, thereby effectively inhibiting tumor angiogenesis and improving the curative effect on the tumors. The new application of the VEGFA signal and FGF-2 signal double-effect inhibitor opens up a new way for treating nasopharyngeal carcinoma or FGF-2 high-expression solid tumors, and has a relatively obvious treatment effect.

The application also provides application of the FGF-2 signaling inhibitor in preparation of a medicine for improving the drug resistance of tumors to the VEGFA signaling inhibitor, wherein the tumors have drug resistance to the VEGFA signaling inhibitor.

In the application, the application of the FGF-2 signaling inhibitor can effectively improve and treat (eliminate) the drug resistance of tumors to the VEGFA signaling inhibitor, so as to optimize the tumor treatment effect of the existing VEGFA signaling inhibitor. Specifically, the FGF-2 signal inhibitor inhibits the expression of downstream signal molecules such as MYC through a receptor of FGF-2 on tumor endothelial cells, so that the proliferation of the vascular endothelial cells is inhibited, the angiogenesis is further hindered, the proliferation of the tumor cells is inhibited, the tumor volume is reduced, the apoptosis of the tumor cells is promoted, and the effect of treating tumors is further achieved.

In an embodiment of the application, the FGF-2 signaling inhibitor inhibits at least one of an FGF-2 ligand, an FGFR1 receptor, and a downstream signaling pathway key molecule MYC thereof, and the FGF-2 signaling inhibitor comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug. The specific type of FGF-2 signaling inhibitor is as described above and will not be described further herein.

In embodiments herein, the VEGFA signaling inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and its downstream signaling pathway key molecule MYC, and the VEGFA signaling inhibitor includes at least one of a chemical drug, a polypeptide drug, a protein drug and a gene drug.

In an embodiment of the present application, the FGF-2 signaling inhibitor is present as a single active ingredient or is present as a pharmaceutical with other pharmaceutically acceptable active ingredients.

In embodiments of the present application, the tumor comprises a solid tumor. In an embodiment of the present application, the solid tumor includes at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroids, and nasopharyngeal carcinoma. In one embodiment, the solid tumor comprises nasopharyngeal carcinoma. FGF-2 signaling inhibitors are effective in treating nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors, and FGF-2 signaling inhibitors are effective in treating nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors. Nasopharyngeal carcinoma is characterized in that tumor cells thereof highly express FGF-2; FGF-2 can activate the expression of downstream signal molecules such as MYC and the like through one of FGFR1 highly expressed in endothelial cells, so that the endothelial cell proliferation and angiogenesis are alternatively activated, and the drug resistance effect of the VEGFA signal inhibitor on angiogenesis cannot be inhibited.

In the present embodiment, the medicament may further include a pharmaceutically acceptable carrier and/or adjuvant. The selection of the carrier and the auxiliary material in the medicine for improving the drug resistance of the tumor to the VEGFA signal inhibitor is as described above; the form, administration mode and dosage of the medicament are described in the above-mentioned medicaments for treating tumors, and are not described herein again.

Nasopharyngeal carcinoma naturally has high expression of FGF-2, and in nasopharyngeal carcinoma or solid tumors with high expression of FGF-2, FGF-2 signals can act on FGFR1 receptors on endothelial cells in tumors, activate downstream signal pathways, promote proliferation of vascular endothelial cells, and maintain angiogenesis in a compensatory manner, so that the drug resistance to a VEGFA signal inhibitor is caused. The FGF-2 signal inhibitor can block the drug resistance mechanism of the tumor, thereby effectively reversing the drug resistance of the tumor to the VEGFA signal inhibitor and improving the curative effect of the VEGFA signal inhibitor on nasopharyngeal carcinoma or FGF-2 high-expression solid tumors. The new application of the FGF-2 signal inhibitor provided by the application can effectively improve the drug resistance of tumors such as nasopharyngeal carcinoma to the VEGFA signal inhibitor and optimize the treatment effect of the existing VEGFA signal inhibitor.

The application also provides a medicament for treating tumors with drug resistance to VEGFA signaling inhibitors, wherein the medicament comprises VEGFA signaling and at least one of FGF-2 signaling double-effect inhibitors and FGF-2 signaling inhibitors.

In an embodiment of the present application, the VEGFA signaling and FGF-2 signaling dual-effect inhibitor comprises lenvatinib.

In one embodiment, the agent for treating a tumor that is resistant to an inhibitor of VEGFA signaling comprises a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling. In another embodiment, the agent for treating a tumor that is resistant to a VEGFA signaling inhibitor comprises an FGF-2 signaling inhibitor and a VEGFA signaling inhibitor.

In the present application, the medicament may further comprise a carrier and/or an adjuvant; the medicament may also include other pharmaceutically acceptable active ingredients. The selection of the carrier and the auxiliary materials is as described above, and the detailed description is omitted here.

The application also provides a medicine for improving the drug resistance of tumors to VEGFA signaling inhibitors, and the medicine comprises FGF-2 signaling inhibitors.

The experimental conditions are presented in this application:

(1) Cell culture

The human nasopharyngeal carcinoma cell lines 5-8F, HONE-1 and CNE1 adopted in the application are provided by Shenzhen second national hospital friendship. The human a549 lung cancer cell line was provided by the university of double denier cell and department of genetic medicine. Human SK-MEL-5 melanoma, hepG2 hepatocellular carcinoma, MDA-MB-231 breast carcinoma, SW480 colon carcinoma, PANC-1 pancreatic ductal adenocarcinoma, 293T embryonic kidney cells, murine T241 fibroma cell line and FGF-2 overexpressed T241 fibroma cell line, murine 4T1 breast cancer cell line and FGF-2 overexpressed 4T1 breast cancer cell line are provided by the Carolina medical institute of Sweden. The cells were cultured in DMEM medium (catalog No. MA0213, meilunbio) or RPMI 1640 medium (catalog No. MA0215, meilunbio) containing 100U/mL of penicillin, 100. Mu.g/mL of streptomycin (catalog No. MA0110, meilunbio) in 10-% FBS (catalog No. 40130ES76, YEASEN), respectively. In the FGF-2 gene shRNA experiment, nasopharyngeal carcinoma tumor cells were stably transfected with lentiviral vectors made from 293T embryonic kidney cells.

(2) Animal experiments

The animal experiments of the present application used 6-8 weeks old male BALB/C-nu/nu nude mice, male C57/B6 mice, or female BALB/C mice, purchased from Jiejicaokang Biotech. Mice were maintained under standard animal feeding conditions and fed a standard diet. When constructing mouse xenograft tumor model and mouse graft tumor model, 1 × 10 suspended in 100 μ LPBS ⁶ 5-8F nasopharyngeal carcinoma cells, T241 fibroma cells or 4T1 breast cancer cells were implanted subcutaneously in nude mice, subcutaneously in C57/B6 mice, or in BALB/C mice mammary fat pads, and the tumor size was measured every other day with calipers and the volume of the transplanted tumor was calculated according to a standard formula.

When the treatment effect of different medicinal preparations on mice of different tumor models is discussed, the medicinal preparations can be administered by intragastric administration or intraperitoneal injection to the mice of different experimental groups. After a period of time, the mice were sacrificed, dissected and examined histologically, RNA and protein extraction, tumor vascular density, etc.

When tumor metastasis is studied, the tumor size is 2.0cm ³ In time, primary tumor resection surgery was performed on the mice. After mice were anesthetized using a anesthesia machine, tumors were surgically excised. Feeding water containing antibiotics, paying attention to the recovery of the body temperature of the mouse, closely observing, independently placing the mouse in a cage for feeding after the normal activity of the mouse is recovered, checking the wound healing and the mouse activity condition every 4-6h, and continuing feeding after the wound is completely healed. 4-6 weeks after completion of subcutaneous tumor resection, mice were sacrificed, dissected and subjected to histological examination, pulmonary metastasis examination, histological examination, and the like.

Experiment 1: VEGFA signaling inhibitor-resistant nasopharyngeal carcinoma-specific high-expression FGF-2 compared to non-drug-resistant colon cancer

Clinical trial results found that colon cancer is less resistant to VEGFA signaling inhibitors. Inhibitors of VEGFA signaling such as bevacizumab have become an important first-line therapy for colon cancer. However, most nasopharyngeal cancers have drug resistance of a VEGFA signal inhibitor, so bevacizumab has poor performance in clinical trials for treating nasopharyngeal cancer. FIG. 1 is a graph showing the comparison of the expression levels of pro-angiogenic factors in various tumors, wherein A is a graph showing the comparison of the expression levels of pro-angiogenic factors in tumor RNA expression profiles, and B is a graph showing the comparison of the relative expression levels of FGF-2 in tumor cell lines. In A of FIG. 1, the RNA expression profiles of large-scale human tumors (clear cell renal carcinoma KIRC/colon carcinoma COAD/nasopharyngeal carcinoma NPC/gastric carcinoma STAD/pancreatic carcinoma PAAD/lung adenocarcinoma LUAD/breast carcinoma BRCA/skin melanoma SKCM /) of various tumor types in TCGA public database and Oncomine database were analyzed by cross-platform alignment, and FGF-2 was specifically expressed in nasopharyngeal carcinoma and expressed in colon carcinoma in various angiogenesis promoting factors (vascular endothelial growth factor VEGFA/human fibroblast growth factor 2 FGF-2/platelet-derived growth factor B PDGFB/epidermal growth factor EGF/angiopoietin 1ANGPT 1/erythropoietin EPO) compared with the respective Control group (Control). Using multiple types of human tumor cell lines in B of FIG. 1, three nasopharyngeal carcinoma (NPC) cell lines were found to express FGF-2 at high levels, whereas human colon carcinoma cells (SW 480) expressed FGF-2 at the lowest level in the test group. Thus, FIG. 1 shows that FGF-2 is expressed at high levels in nasopharyngeal carcinoma.

Experiment 2: the drug resistance of nasopharyngeal carcinoma tumor-bearing mice to the VEGFA signal inhibitor can be improved by knocking down FGF-2 gene in nasopharyngeal carcinoma cells

Constructing 5-8F nasopharyngeal carcinoma tumor-bearing mice, and increasing the tumor to 0.5cm ³ At this time, mice were randomly divided into two groups, one experimental group, treated with VEGFA signaling inhibitor (bevacizumab) (Anti-VEGF), and the other control group, treated with PBS buffer (Vehicle). Each group was injected intraperitoneally at a concentration of 2.5mg/kg, 2 times per week. FIG. 2 shows the experimental results of drug resistance of nasopharyngeal carcinoma tumor-bearing mice to VEGFA signal inhibitor, wherein A is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells, B is the tumor volume of nasopharyngeal carcinoma tumor-bearing mice transplanted with 5-8F nasopharyngeal carcinoma cells knocked down with FGF-2 gene, C is the tumor vascular density result, and D is the tumor lung metastasis result.

FIG. 2A shows that the nasopharyngeal carcinoma tumor-bearing mice (NPC-shScrambled) transplanted with 5-8F nasopharyngeal carcinoma cells are resistant to VEGFA signaling inhibitor, and the tumor volumes of the experimental group and the control group are not significantly different, i.e. there is no difference in tumor growth between the two groups. The same experiment as described above was carried out using 5-8F nasopharyngeal carcinoma cells with the FGF-2 gene knocked-down in B of fig. 2, and it can be seen that a nasopharyngeal carcinoma tumor-bearing mouse (NPC-shFGF 2) into which 5-8F nasopharyngeal carcinoma cells with the FGF-2 gene knocked-down were transplanted was not resistant to VEGFA signaling inhibitors, and tumor growth was significantly inhibited by VEGFA signaling inhibitors.

Further, it can be seen in C of fig. 2 that in the control nasopharyngeal carcinoma tumors, the vascular density was not affected by the VEGFA signaling inhibitor. In the nasopharyngeal carcinoma tumor with FGF-2 gene knocked down, the same medicine and the same condition are used for treatment, and the vascular density is obviously inhibited.

Furthermore, in fig. 2D, tumor metastasis experiments were performed using the same tumor-bearing mice as described above, and it was found that in the control group of nasopharyngeal carcinoma tumors, the incidence of lung metastasis was not significantly affected by VEGFA signaling inhibitors; and in the nasopharyngeal carcinoma tumor with the FGF-2 gene knocked down, the incidence of lung metastasis is obviously inhibited by using the same medicine and the same condition for treatment. Thus, figure 2 demonstrates that blocking FGF-2 signaling can improve resistance of nasopharyngeal carcinoma to inhibitors of VEGFA signaling.

Experiment 3: other types of tumors that highly express FGF-2 can increase tumor growth, vascular density, and metastasis

Tumor-bearing mice of the T241 fibroma of the control group and the T241 fibroma of the high expression FGF-2 are constructed, and are respectively a T241-Vector group and a T241-FGF-2 group. Tumor-bearing mice for constructing the 4T1 breast cancer of the control group and the 4T1 breast cancer with high expression of FGF-2 are respectively a 4T1-Vector group and a 4T1-FGF-2 group. The results of the research on the effects of FGF-2 in fibroma and breast cancer are shown in fig. 3, where a is the tumor volume of the fibroma-bearing mouse, B is the tumor vascular density result of the fibroma-bearing mouse, C is the tumor lung metastasis result in the fibroma-bearing mouse, D is the tumor volume of the breast cancer-bearing mouse, E is the tumor vascular density result of the breast cancer-bearing mouse, and F is the tumor lung metastasis result in the breast cancer-bearing mouse.

As can be seen from a-C of fig. 3, tumor growth was significantly enhanced and vascular density was significantly increased using T241 fibroma cells highly expressing the FGF-2 gene; the tumor metastasis experiment carried out by utilizing tumor-bearing mice shows that the incidence rate of lung metastasis is obviously enhanced. As can be seen from D-F of fig. 3, tumor growth was significantly enhanced and vascular density was significantly increased using 4T1 breast cancer cells that highly express the FGF-2 gene; tumor metastasis experiments are carried out by using tumor-bearing mice, so that the incidence rate of lung metastasis is obviously enhanced. FIG. 3 shows that in other tumors than nasopharyngeal carcinoma, high expression of FGF-2 signaling can enhance angiogenesis, thus indicating that the mechanism of experiment 1 can also occur in other tumor types that highly express FGF-2 signaling.

Experiment 4: VEGFA signal and FGF-2 signal double-effect inhibitor can treat nasopharyngeal carcinoma tumor-bearing mice resistant to VEGFA signal inhibitor

Constructing 5-8F nasopharyngeal carcinoma tumor-bearing mice, and increasing the tumor to 0.5cm ³ When the mice are randomly divided into three groups, one group is an experimental group, and the mice are treated by adopting VEGFA signals and FGF-2 signal double-effect inhibitors (Lunvatinib); one group was a control group treated with VEGFA signaling inhibitor (bevacizumab) (Anti-VEGF); one group was blank and treated with solvent (Vehicle). Wherein bevacizumab is intraperitoneally injected at a concentration of 2.5mg/kg, 2 times per week; the results of studies on the effects of the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling were shown in fig. 4, where a is tumor volume, B is tumor vascular density, and C is tumor lung metastasis, with the gavagainb administered at a concentration of 30mg/kg 1 time per day (earlier experiments showed that bevacizumab and ranvatinib at the above concentrations had consistent efficacy on non-drug resistant colon cancer).

In a of fig. 4, it can be seen that the nasopharyngeal carcinoma tumor-bearing mice did not respond consistently to VEGFA signaling and FGF-2 signaling dual-effect inhibitors, and VEGFA signaling inhibitors, which were resistant to VEGFA signaling inhibitors (bevacizumab), but had strong responses to VEGFA signaling and FGF-2 signaling dual-effect inhibitors (ranvatinib), indicating that VEGFA signaling and FGF-2 signaling dual-effect inhibitors effectively treated nasopharyngeal carcinoma resistant to VEGFA signaling inhibitors.

Further, it can be seen in B of fig. 4 that vascular density in nasopharyngeal carcinoma tumors was not affected by VEGFA signaling inhibitors; in the VEGFA signal and FGF-2 signal double-effect inhibitor treatment group, the blood vessel density is obviously inhibited.

Further, in fig. 4C, tumor metastasis experiments were performed using the same tumor-bearing mice, and it was found that in nasopharyngeal carcinoma tumors, the incidence of lung metastasis was not significantly affected by VEGFA signaling inhibitors; and in the VEGFA signal and FGF-2 signal double-effect inhibitor treatment group, the incidence rate of lung metastasis is remarkably inhibited. Thus, experiment 4 shows that the use of a dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is effective in treating nasopharyngeal carcinoma.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of a VEGFA signalling and a dual effect inhibitor of FGF-2 signalling and/or an inhibitor of FGF-2 signalling for the preparation of a medicament for the treatment of tumours which are resistant to VEGFA signalling inhibitors.
2. The use of claim 1, wherein said dual-effect inhibitor of VEGFA signaling and FGF-2 signaling is capable of inhibiting both the VEGFA signaling pathway and the FGF-2 signaling pathway of endothelial cells in said tumor, thereby overcoming resistance of said tumor to said inhibitor of VEGFA signaling and inhibiting tumor angiogenesis.
3. The use of claim 1, wherein the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises ranvatinib;

the FGF-2 signal inhibitor inhibits at least one of FGF-2 ligand, FGFR1 receptor and downstream signaling pathway key molecule MYC, and the FGF-2 signal inhibitor comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and downstream signal pathway key molecule MYC, and comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

the VEGFA signal and FGF-2 signal double-effect inhibitor is used as a single active ingredient or forms the medicament with other pharmaceutically acceptable active ingredients;

the FGF-2 signaling inhibitor is used as a single active ingredient or forms the medicament together with other pharmaceutically acceptable active ingredients.
4. The use of claim 1, wherein the tumor comprises a solid tumor comprising at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroids, and nasopharyngeal cancer; further, the solid tumor includes nasopharyngeal carcinoma;

the tumor is a solid tumor expressing FGF-2.
Use of an FGF-2 signalling inhibitor for the preparation of a medicament for improving the resistance of a tumour to a VEGFA signalling inhibitor, said tumour being resistant to a VEGFA signalling inhibitor.
6. The use of claim 5, wherein the FGF-2 signaling inhibitor inhibits at least one of an FGF-2 ligand, an FGFR1 receptor, and a downstream signaling pathway key molecule MYC thereof, and wherein the FGF-2 signaling inhibitor comprises at least one of a chemical drug, a polypeptide drug, a protein drug, and a gene drug;

the VEGFA signal inhibitor inhibits at least one of VEGFA ligand, VEGFR2 receptor and downstream signal pathway key molecule MYC, and comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs;

the FGF-2 signaling inhibitor is used as a single active ingredient or forms the medicament together with other pharmaceutically acceptable active ingredients.
7. The use of claim 5, wherein the tumor comprises a solid tumor comprising at least one of liver cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, esophageal cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, cervical cancer, renal clear cell carcinoma, skin melanoma, fibroids, and nasopharyngeal cancer; further, the solid tumor includes nasopharyngeal carcinoma;

the tumor is a solid tumor expressing FGF-2.
8. A medicament for treating a tumor that is resistant to a VEGFA signaling inhibitor, comprising VEGFA signaling and at least one of a dual-effect inhibitor of FGF-2 signaling and an inhibitor of FGF-2 signaling.
9. The medicament of claim 8, wherein the dual-effect inhibitor of VEGFA signaling and FGF-2 signaling comprises ranvatinib.
10. A medicament for improving the resistance of a tumour to VEGFA signalling inhibitors, characterised in that the medicament comprises an FGF-2 signalling inhibitor.