Application of a protein polypeptide in inhibiting malignant ascites and malignant pleural effusion

Abstract

The present invention discloses an application of a protein polypeptide in inhibiting malignant ascites and malignant pleural effusion, and relates to the field of treatment of malignant ascites and malignant pleural effusion. The present invention injects the protein polypeptides shown in SEQ ID NO.01 and SEQ ID NO.02 into an ascites model, and finds that the production of malignant pleural effusion and malignant ascites can be inhibited, the amount of malignant pleural effusion and malignant ascites can be reduced, and the survival time can be prolonged. This application method of the protein polypeptide is the first discovery in the industry, and provides a theoretical and practical basis for the development of drugs for the clinical treatment of malignant pleural effusion and malignant ascites.

Description

Technical Field

[0001] The invention relates to the field of treatment of malignant ascites and malignant pleural effusion, in particular to a protein polypeptide for inhibiting malignant ascites and malignant pleural effusion. Background Art

[0002] Malignant ascites is a clinical manifestation of many late-stage malignant tumors. Usually, this occurs in the late stage of the disease. The appearance of malignant ascites is often accompanied by distant metastasis of malignant tumors, at which time drug treatment has become difficult. Therefore, the formation of ascites indicates that there is usually a lack of effective treatment methods in clinical practice. Ascites formation is particularly prominent in patients with advanced ovarian, pancreatic and gastrointestinal malignancies. At this stage, clinical treatments mainly include: simple ascites extraction, intraperitoneal injection of chemotherapy drugs and intraperitoneal hyperthermic perfusion therapy, etc. However, the above methods are often accompanied by greater toxic effects. Therefore, the development of drugs with significant effects and low side effects shows a wider market demand and application progress.

[0003] Malignant pleural effusion (MPE) is one of the most common complications of advanced metastatic cancer. It is caused by pleural metastasis and is characterized by the presence of malignant cells. Tumor cells infiltrate the pleural cavity through direct invasion, hematogenous metastasis, or lymphatic spread. Pleural effusion may be the result of tumor growth blocking lymphatic drainage.

[0004] MPE can occur in 15% of cancer patients, the most common of which is lung cancer (LC), with an incidence of up to 60%, followed by breast cancer (BC), lymphoma, gynecological tumors and malignant mesothelioma. The average survival (OS) of patients after the initial diagnosis of MPE is 3-12 months. Common symptoms of most MPE patients include dyspnea, cough and chest pain. The severity of the accompanying symptoms depends on the amount or volume of effusion and the patient's cardiopulmonary status. In recent years, with the in-depth study of the pathogenesis of MPE, clinical treatment methods have been improved. At present, there are many clinical treatment methods for MPE, but the overall treatment efficiency is low. Among them, intrathoracic infusion of chemotherapy drugs for the treatment of MPE is the most widely used method, but its efficacy is not satisfactory, and long-term toxic side effects (e.g., bone marrow suppression, gastrointestinal reactions, fever, chest pain and pleural fibrosis, etc.) are large.

[0005] The Chinese invention patent 2021111220700, which was previously applied for and not yet published by the applicant of the present invention, provides a polypeptide combination for inhibiting tumor invasion and metastasis and its application, and its working principle is to provide new therapeutic drugs for the clinical treatment of patients with tumor metastasis by inhibiting the TGF-β / SMADs signaling pathway. The applicant also published a foreign paper "EZH2-triggeredmethylation of SMAD3 promotes its activation and tumor metastasis" (Changsheng Huang, The Journal of Clinical Investigation, doi: 10.1172 / JCI152394), which disclosed that the methylation of lysine at positions 53 and 333 of SMAD3 can affect its phosphorylation activation and can affect the expression of marker proteins of epithelial-to-cytoplasmic transformation in its tumor cells. However, there are few relevant literature reports on how to inhibit malignant ascites and malignant pleural effusion. In the prior art, Chinese medicine compositions are generally used to treat malignant ascites, such as the Chinese medicine prescriptions for treating hepatitis B, liver cirrhosis, ascites, and tumors provided in Chinese patent application CN113288999A; there are also a small number of polypeptide drugs, such as Chinese patent application CN106999539A, which uses polypeptide drugs (terlipressin or its salt) as continuous infusion as a treatment method for patients with ascites. The document "Observation on the Efficacy of Hepatic Hydrolysis Peptides in Treating Cirrhotic Ascites" (Li Huimei, Master's thesis of Jilin University, 2007) discloses that the use of hepatic hydrolysis peptides (PHGF) (low molecular weight polypeptides extracted from fresh liver cells of suckling pigs) can significantly increase the ascites regression rate of patients with cirrhosis. The document "Observation on the Efficacy of Hepatic Hydrolyzed Peptides in Treating Ascites in Hepatitis B Cirrhosis" (Ciren, "Highland Medicine", Issue 7, 2017) also disclosed that the use of liver hydrolyzed peptide injection from Hunan Wuzhoutong Pharmaceutical Co., Ltd., compared with the injection of reduced glutathione, the increase in serum albumin was more obvious, and the number of days for the ascites to subside was shorter than that of the control group. It can be seen that the use of protein peptides to treat ascites is relatively rare in the industry. Summary of the invention

[0006] In view of the above deficiencies in the prior art, the present invention provides an application of a protein polypeptide in inhibiting malignant ascites and malignant pleural effusion, and applies the protein polypeptide provided in the prior patent application (application number 2021111220700) to the inhibition and treatment of malignant pleural effusion and malignant ascites, which has the advantages of simple and easy-to-obtain raw materials, high drug efficiency, low side effects, not easy to accumulate poisoning, and relatively specific targets, and has a very high application prospect. It is specifically achieved through the following technologies.

[0007] A protein polypeptide is used in the preparation of a drug for inhibiting malignant ascites and malignant pleural effusion. The protein polypeptide comprises two polypeptides, the first polypeptide comprises a cell-penetrating peptide I and a targeting peptide P1, and the second polypeptide comprises a cell-penetrating peptide II and a targeting peptide P2; the amino acid sequence of the targeting peptide P1 is shown in SEQ ID NO.01, and the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO.02.

[0008] The therapeutic polypeptide involved in the present invention can produce a mild inflammatory reaction locally through local administration, achieve closure of pleural and peritoneal adhesions, activate the local immune ability of the body, and then inhibit the development of malignant pleural effusion and malignant ascites. It should be noted that the cell-penetrating peptide I and cell-penetrating peptide II can adopt any amino acid sequence existing in the prior art, as long as they can achieve the inherent function of the cell-penetrating peptide, they can be applied in the present invention. The amino acid sequences of the cell-penetrating peptide I and the cell-penetrating peptide II in the present invention can be the same or different.

[0009] The mechanism of action of the inhibitory treatment of tumor invasion and metastasis is fundamentally different from the treatment of pleural effusion and ascites. In terms of the treatment of malignant pleural effusion and malignant ascites, the therapeutic polypeptide involved in the present invention can inhibit the body's immune function through local administration, and more importantly, it can produce a mild inflammatory response locally to achieve closure of pleural and peritoneal adhesions. Therefore, this kind of polypeptide can activate the body's local immune ability, thereby inhibiting the further occurrence and development of tumors. In addition, this kind of polypeptide can inhibit the local dissemination of tumor cells in the pleural cavity and peritoneal cavity. Therefore, how to inhibit tumor invasion and metastasis is not directly related to how to relieve malignant ascites.

[0010] Preferably, the amino acid sequences of the cell-penetrating peptide I and the cell-penetrating peptide II are both as shown in SEQ ID NO.03.

[0011] Preferably, the amino acid sequences of the two polypeptides are shown as SEQ ID NO.04 and SEQ ID NO.05, respectively.

[0012] Preferably, the medicine is an intravenous injection containing the protein polypeptide.

[0013] More preferably, in the drug, the concentration of the protein polypeptide is 2.5-3.5 mg / mL. Generally speaking, the specification of the drug is 100 μL.

[0014] The production of malignant pleural effusions and ascites is mostly caused by tumor cells directly infiltrating or distant tumor cell metastasis and colonization in the pleural cavity or peritoneal cavity. Because tumor cells invade and block local capillaries and lymphatic vessels, malignant pleural effusions and ascites often contain a large amount of protein and blood. The production of other common pleural effusions and ascites (such as exudative pleural effusions and ascites, exudative pleural effusions and ascites, and tuberculous pleural effusions and ascites, etc.) is not caused by local invasion of tumor cells or distant colonization and metastasis. The applicant, through in-depth research on protein polypeptides, first constructed a malignant ascites and malignant pleural effusion mouse model, and then injected protein polypeptides in the peritoneal cavity, and found that the injection of the above-mentioned protein polypeptides can inhibit the production of ascites and malignant pleural effusions, and reduce the amount of malignant ascites and pleural effusions.

[0015] Compared with the prior art, the present invention is beneficial in that: the present invention provides a therapeutic drug that can treat malignant ascites and malignant pleural effusion, namely, the protein polypeptide provided by the present invention. The use of the protein polypeptide in treating malignant ascites and malignant pleural effusion is the first discovery in the industry, and can provide a theoretical and practical basis for the development of drugs for the clinical treatment of malignant ascites and malignant pleural effusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The changes in abdominal circumference of mice with malignant ascites in each group in Experimental Example 1;

[0017] Figure 2 These are the imaging images of mice with malignant ascites in each group in Experimental Example 1;

[0018] Figure 3 The content of malignant ascites in each group of mice with malignant ascites in Experimental Example 1;

[0019] Figure 4 The survival curves of each group of mice with malignant ascites in Experimental Example 1;

[0020] Figure 5 These are the imaging images of mice with malignant pleural effusion in each group in Experimental Example 2;

[0021] Figure 6 is the content of malignant pleural effusion in each group of mice in Experimental Example 2;

[0022] Figure 7 The survival curves of mice with malignant pleural effusion in each group of Experimental Example 2;

[0023] Figure 8 Schematic diagram of the amino acid residue sequence of the protein polypeptide used in Experimental Examples 1 and 2. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] In the following experimental examples, the amino acid sequences of the two polypeptides of the protein polypeptide used are shown in SEQ ID NO.04 and SEQ ID NO.05 respectively, and the abbreviation diagram of the amino acid sequence is shown in the attached Figure 8 As shown; they were synthesized separately by entrusting Wuhan Huamei Bioengineering Co., Ltd. The two polypeptides were made into 1 μg / mL intravenous injection solution using PBS buffer.

[0026] The amino acid sequence of the first polypeptide is: Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg ArgAla Leu Asp Glu Leu Glu Lys Ala Ile Thr Thr Gln;

[0027] The amino acid sequence of the second polypeptide is: Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg ArgAla Pro Ala Thr Val Cys Lys IIe Pro Pro Gly Cys;

[0028] In the above two polypeptide sequences, the amino acid sequence of the membrane-penetrating peptide is: Tyr Gly Arg Lys Lys Arg ArgGln Arg Arg Arg Ala.

[0029] Experimental Example 1: Effect of peptide drugs on the occurrence and development of malignant ascites

[0030] 1. Construction of malignant ascites model

[0031] 1×10 6 CT26 BALB / c colon cancer cell lines (purchased from The Global Bioresource Center cell bank in the United States) were injected into the abdominal cavity of 60 8-week-old BALB / c mice from the right lower abdomen of mice, and the mice were randomly divided into three groups: A, B, and C. Among them, 10 mice in each group were observed for changes in abdominal circumference and ascites content, and the remaining 10 mice were observed for survival time.

[0032] 2. Effect of intraperitoneal injection of peptide drugs on malignant ascites

[0033] The protein peptide drugs required for this experimental example were prepared by Wuhan Huamei Bioengineering Co., Ltd. One day after the malignant ascites model was established, mice in group A were injected with 100 μL of PBS solution into the peritoneal cavity every two days; mice in group B were injected with 5-fu chemotherapy drugs (25 mg / kg; 100 μL) into the peritoneal cavity every two days; mice in group C were injected with protein peptide drugs (1 mg / ml; 100 μL) into the peritoneal cavity every two days; after medication, the abdominal circumference of the mice was measured every five days starting from the second day, and the results are shown in the attached Figure 1 .from Figure 1 It can be seen that the abdominal circumference of mice using 5-fu chemotherapy drugs and peptide drugs was significantly smaller than that of mice using PBS group.

[0034] On the 16th day after medication, the growth of abdominal tumors in mice was observed by small animal in vivo imaging. Figure 2 .from Figure 2 It can be seen that the number of peritoneal cells in mice using 5-fu chemotherapy drugs and peptide drugs was significantly less than that in the mice using PBS group.

[0035] On the 16th day after administration, 10 mice in each group were randomly killed, and the ascites content was collected and statistically tested by Student t test. The results are shown in the attached Figure 3 .from Figure 3 It can be seen that the amount of ascites in mice treated with 5-fu chemotherapy and peptide drugs was significantly smaller than that in mice treated with PBS.

[0036] After the malignant ascites model was established, the survival time of the mice was observed over time, and the survival curve of the mice was drawn. The results are shown in the attached Figure 4 .from Figure 4 It can be seen that the survival time of mice using 5-fu chemotherapy drugs and peptide drugs was significantly longer than that of mice using PBS group.

[0037] From the above experiments, it can be found that the protein polypeptide of the present invention can effectively inhibit the development of malignant ascites.

[0038] Experimental Example 2: Effect of peptide drugs on the occurrence and development of malignant pleural effusion

[0039] 1. Construction of malignant pleural effusion model

[0040] 1×10 6 Lewis C57 lung cancer cell lines (purchased from the Global Bioresource Center cell bank in the United States) were injected into the chest cavity of 60 8-week-old C57 mice through the right lower rib space of the mice, and the mice were randomly divided into three groups: D, E, and F. Among them, 10 mice in each group were observed for changes in abdominal circumference and ascites content, and the remaining 10 mice were observed for survival time.

[0041] 2. Effect of intrathoracic injection of peptide drugs on malignant pleural effusion

[0042] The protein peptide drugs required for this experimental example were prepared by Wuhan Huamei Bioengineering Co., Ltd. One day after the malignant ascites model was established, mice in group D were injected with 100 μL of PBS solution into the right thoracic cavity every two days; mice in group E were injected with cisplatin chemotherapy drugs (1 mg / kg; 100 μL) into the peritoneal cavity every two days; mice in group F were injected with protein peptide drugs (1 mg / ml; 100 μL) into the peritoneal cavity every two days; on the sixteenth day after medication, the growth of mouse abdominal tumors was observed by small animal live imaging, see attached Figure 5 .from Figure 5 It can be seen that the number of peritoneal cells in mice using cisplatin chemotherapy and peptide drugs was significantly less than that in the mice using PBS.

[0043] On the 16th day after administration, 10 mice in each group were killed, and the pleural effusion content was collected and statistically tested by Student t. The results are shown in the attached Figure 6 .from Figure 6 It can be seen that the amount of pleural effusion in mice using cisplatin chemotherapy and peptide drugs was significantly smaller than that in the mice using PBS.

[0044] After the malignant pleural effusion model was established, the survival time of the mice was observed over time, and the survival curve of the mice was drawn. The results are shown in the attached Figure 7 .from Figure 7 It can be seen that the survival time of mice using cisplatin chemotherapy and peptide drugs was significantly longer than that of mice using PBS.

[0045] From the above experiments, it can be found that the protein polypeptide of the present invention can effectively inhibit the development of malignant pleural effusion.

[0046] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Use of a protein polypeptide in the preparation of a drug for treating malignant ascites and malignant pleural effusion, characterized in that: The protein polypeptide consists of two polypeptides, the first polypeptide consists of a cell-penetrating peptide I and a targeting peptide P1, and the second polypeptide consists of a cell-penetrating peptide II and a targeting peptide P2; the amino acid sequence of the targeting peptide P1 is shown in SEQ ID NO.01, and the amino acid sequence of the targeting peptide P2 is shown in SEQ ID NO.02; the amino acid sequences of the cell-penetrating peptide I and the cell-penetrating peptide II are both shown in SEQ ID NO.

03.

2. The use of the protein polypeptide according to claim 1 in the preparation of a drug for treating malignant ascites and malignant pleural effusion, characterized in that: The amino acid sequences of the two polypeptides are shown in SEQ ID NO.04 and SEQ ID NO.05 respectively.

3. Use of the protein polypeptide according to claim 1 in the preparation of a drug for treating malignant ascites and malignant pleural effusion, characterized in that: The medicine is an intravenous injection containing the protein polypeptide.

4. Use of the protein polypeptide according to claim 3 in the preparation of a drug for treating malignant ascites and malignant pleural effusion, characterized in that: In the medicine, the concentration of the protein polypeptide is 2.5-3.5 mg / mL.

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