A small molecule polypeptide TCMP-Y1 labeled with iodine-131 and its preparation method and application

Abstract

The present invention discloses a small molecule polypeptide TCMP-Y1 labeled with iodine-131, and a preparation method and application thereof. TCMP-Y1 is a small molecule polypeptide separated and identified by the inventor from toad skin secretions in the early stage, and its amino acid sequence is YFKAWGWPIDDATTEHL-NH2. TCMP-Y1 polypeptide has no obvious proliferation inhibition effect on prostate cancer cells, but iodine-131 labeled 131 I‑TCMP‑Y1 can bind to prostate cancer cells and stably retain them. It also inhibits tumor cell proliferation and tumor growth in vitro and in vivo. It is water-soluble 131 I-TCMP-Y1 is mainly excreted through the kidney and urinary system, thus reducing the risk of radiation-induced liver injury. TCMP-Y1 is a small molecule peptide that is good at introducing radionuclides into the body, improving drug safety and tumor targeting, and can be used as a potential drug for tumor intravenous irradiation therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to an iodine-131 labeled small molecule polypeptide TCMP-Y1 and a preparation method and application thereof. Background Art

[0002] Prostate cancer seriously threatens the life and health of men. Its morbidity and mortality rate rank second and fifth among men in the world, respectively. The main treatments for early prostate cancer or localized prostate cancer are radical surgery and androgen deprivation therapy (ADT), which can often achieve a cure. However, with the further development of prostate cancer, almost all prostate cancer patients who are sensitive to androgen deprivation therapy in the early stages of the disease will develop hormone resistance, namely castration-resistant prostate cancer (CRPC). Related studies have pointed out that due to the insidious onset of prostate cancer, the lack of specificity of early symptoms, and the lack of effective screening programs for prostate cancer in my country, most patients have already developed to the late stage of prostate cancer when they feel unwell and seek medical treatment or even when they are diagnosed, missing the best time for surgical treatment. Therefore, it is urgent to develop new treatments to treat prostate cancer in clinical practice.

[0003] Radiotherapy is an indispensable method in the treatment of prostate cancer. Most prostate cancer patients need radiotherapy, which includes external radiation therapy and internal radiation therapy (IRT). IRT uses the characteristics of radionuclides or their labeled compounds that can be targeted and aggregated in diseased tissues. Radionuclides are brought into the human body and the α and β rays emitted by radionuclides are used to produce sufficient ionizing radiation biological effects in the lesions, thereby inhibiting or destroying the tissues in the diseased area, leading to metabolic disorders, loss of reproductive capacity, cell aging and even apoptosis of the diseased tissue cells. The α and β rays emitted by radionuclides have high energy and short range. The key to its treatment is to achieve a higher therapeutic effect by maximizing the damage to the tumor while protecting healthy tissues.

[0004] Since the development of nuclear medicine, many kinds of mature radioactive drugs have been developed for treatment, including 131 I - Treat thyroid cancer, 89 Sr treats bone metastases and relieves bone pain. 32 PTreatment of polycythemia vera, 131 I-MIBG treatment of pheochromocytoma, 211At-tamoxifen for the treatment of breast cancer, etc. In recent years, radionuclide-labeled peptides have become a hot trend. Their advantage is that they can be quickly cleared from plasma and enriched in target tissues, thus making their bone marrow toxicity low. For example, octreotide, an analog of somatostatin, is used to label peptides. 111 In-DTPA-octreotide can be used to treat a variety of octreotide receptor-positive tumors.

[0005] Host defense peptides (HDPs) from natural sources, especially antimicrobial peptides (AMPs), have attracted much attention. Antimicrobial peptides are antibacterial, antiviral, antifungal and anticancer by destroying the membrane integrity of bacteria, viruses and fungi or inhibiting certain cell functions. It is worth noting that among the 2981 databases on AMPs, more than 1000 have amphibians as hosts, while only 200 have humans as hosts. A variety of host defense peptides have been isolated from the skin gland secretions of amphibians, such as Australian tree frogs and toads, and most of these peptides have anti-tumor and antibacterial effects. The host defense peptides Caerin 1.1 and Caerin 1.9 peptides isolated from the gland secretions of Australian tree frogs (Litoria genus) have been shown to have multiple biological activities, such as antibacterial, antiviral, antifungal, anticancer and immunomodulatory. The inventor's research team has previously demonstrated that at a certain concentration, Caerin1.1 polypeptide and Caerin1.9 polypeptide have inhibitory effects on a variety of human tumor cells, including cervical cancer cells, breast cancer cells, and anaplastic thyroid cancer cells. Regarding Caerin1.1 polypeptide, Caerin1.9 polypeptide and their iodine-131 markers: 131 I-Caerin1.1 and 131 The effect of I-Caerin1.9 on prostate cancer has not been reported yet. In addition, it is of great significance to develop and explore more host defense peptides with anti-tumor effects from natural sources. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a small molecule polypeptide TCMP-Y1.

[0007] The second object of the present invention is to provide a small molecule polypeptide TCMP-Y1 labeled with iodine-131.

[0008] The third object of the present invention is to provide a method for preparing the iodine-131 labeled small molecule polypeptide TCMP-Y1.

[0009] The fourth object of the present invention is to provide the use of the iodine-131 labeled small molecule polypeptide TCMP-Y1 in the preparation of drugs for treating tumors.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] Previous studies have shown that Caerin1.1 peptide and Caerin1.9 peptide have inhibitory effects on various tumors such as lung cancer and melanoma, and iodine-131 labeled 131 I-Caerin1.1 peptide, 131 I-Caerin1.9 peptide has stronger anti-tumor activity, Caerin1.1 peptide, Caerin1.9 peptide and its iodine-131 marker: 131 I-Caerin1.1 and 131 The effect of I-Caerin1.9 on prostate cancer has not been reported yet. TCMP-Y1 polypeptide is a small molecule polypeptide isolated and identified from toad skin secretions. It is a polypeptide derived from amphibians, just like Caerin1.1 and Caerin1.9. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The therapeutic effect of I-TCMP-Y1 in prostate cancer cell (DU145) transplanted tumors was investigated and compared. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has an inhibitory effect on the proliferation of prostate cancer cells DU145 in vitro and its therapeutic value for prostate cancer in vivo.

[0012] Specifically: (1) The inhibitory effects of Caerin1.1 peptide, Caerin1.9 peptide and TCMP-Y1 peptide on prostate cancer cells (DU145) were investigated by MTT assay and the median inhibition concentration (IC) was determined. 50 ); The anti-tumor effects of Caerin1.1 and Caerin1.9 peptides were verified using plate clone formation experiments.

[0013] (2) Preparation by Chloramine-T (Ch-T) labeling method 131 I-Caerin1.1 peptide, 131 I-Caerin1.9 peptide and 131 I-TCMP-Y1 polypeptide, and its labeling rate, lipid-water partition coefficient and stability were determined.

[0014] (3) Comparison of whether prostate cancer cell DU145 can take up 131I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 and their retention in cells. CCK8 cell proliferation toxicity assay was used to verify 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Different killing effects of I-TCMP-Y1 on prostate cancer cells DU145 in vitro.

[0015] (4) Establish a prostate cancer cell (DU145 cell line) tumor-bearing nude mouse model. 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1, Caerin1.1 peptide, Caerin1.9 peptide and TCMP-Y1 peptide were injected into the tumor to compare their anti-tumor effects in vivo.

[0016] The results showed that: (1) Caerin1.1 peptide and Caerin1.9 peptide both had a significant inhibitory effect on the proliferation of prostate cancer cells, and this effect was enhanced with increasing concentration. The IC 50 They were 10.87μg / mL and 14.50μg / mL respectively; however, TCMP-Y1 polypeptide had no significant inhibitory effect on the proliferation of prostate cancer cells.

[0017] (2) using the chloramine-T labeling method, the labeling rate of the iodine-131 labeled product obtained is greater than 90%; and 131 I-Caerin1.1, 131 I-Caerin1.9 is fat-soluble, 131 I-TCMP-Y1 is water-soluble. The three have high stability in different in vitro environments. 131 I-Caerin1.1, 131 I-Caerin1.9 is more stable.

[0018] (3) Cell uptake and elution experiments prove 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1 can be taken up by DU145 cells and remain relatively stable in the cells. Compared with the simple polypeptide under in vivo or in vitro conditions, 131 I-Caerin1.1, 131 I-Caerin1.9, 131I-TCMP-Y1 has better anti-tumor cell proliferation and tumor growth inhibition capabilities.

[0019] The present invention shows that 131 I-Caerin1.1 peptide, 131 I-Caerin1.9 peptide and 131 I-TCMP-Y1 polypeptide can bind to prostate cancer cells and stably retain them, and inhibit tumor cell proliferation and tumor growth both in vivo and in vitro. 131 I-TCMP-Y1 is mainly excreted through the kidney and urinary system, thereby reducing the risk of radiation-induced liver damage. The present invention provides Caerin1.1 polypeptide, Caerin1.9 polypeptide and iodine-131 labeled polypeptides thereof. 131 I-Caerin1.1 peptide, 131 The invention discloses a new use of I-Caerin1.9 polypeptide in the treatment of prostate cancer and also provides a new small molecule polypeptide TCMP-Y1.

[0020] Specifically, the amino acid sequence of the small molecule polypeptide TCMP-Y1 is YFKAWGWPIDDATTEHL-NH2, as shown in SEQ ID No. 1. Based on the knowledge of the amino acid sequence, those skilled in the art can prepare it by conventional polypeptide synthesis techniques in the art.

[0021] Furthermore, the iodine-131 labeled small molecule polypeptide TCMP-Y1 is a small molecule polypeptide TCMP-Y1 shown in SEQ ID No. 1, wherein radioactive labeled iodine-131 is bound to the amino acid group.

[0022] The present invention also provides a method for preparing the iodine-131 labeled small molecule polypeptide TCMP-Y1, which comprises using a chloramine-T method to prepare a small molecule polypeptide TCMP-Y1 solution, Na- 131 I solution and chloramine-T solution were shaken and mixed at room temperature, and then separated by chromatography. 131 The small molecule polypeptide TCMP-Y1 labeled with I is obtained.

[0023] Furthermore, the TCMP-Y1 and Na- 131 The ratio of I is 40μg:1mCi. 131 I has the highest labeling rate.

[0024] Furthermore, the mass concentration of the TCMP-Y1 solution is 1 mg / mL, and the Na- 131 The ionizing radiation of solution I is 1 mCi (3.7×10 7 Bq); the chloramine-T solution must be prepared and used immediately, with a mass concentration of 1 mg / mL.

[0025] The present invention also provides the use of the iodine-131 labeled small molecule polypeptide TCMP-Y1 in the preparation of drugs for treating tumors, specifically the use of the iodine-131 labeled small molecule polypeptide TCMP-Y1 in the preparation of drugs for intratumoral irradiation treatment.

[0026] Preferably, the tumor includes but is not limited to prostate cancer.

[0027] The present invention also provides a tumor treatment drug, which contains the small molecule polypeptide TCMP-Y1 labeled with iodine-131.

[0028] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0029] The dosage of the drug of the present invention depends on many factors, such as the nature and severity of the disease to be treated, the sex, age, weight and individual response of the patient or animal, the route of administration and the number of times of administration, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms. The dosage level must be selected according to the specific route of administration, the severity of the condition to be treated, and the condition and previous medical history of the patient to be treated. However, it should be recognized that the total daily dosage of the drug of the present invention must be determined by the attending physician within the scope of reliable medical judgment. For any specific patient, the specific therapeutically effective dosage level must be determined according to a variety of factors, including the disorder to be treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate; the duration of treatment; drugs used in combination or simultaneously; and similar factors known in the medical field. For example, the practice in the art is to start the administration of a dose lower than the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained.

[0030] The present invention also provides the use of a Caerin1.1 polypeptide or an iodine-131 labeled Caerin1.1 polypeptide in the preparation of a drug for treating prostate cancer, characterized in that the amino acid sequence of the Caerin1.1 polypeptide is GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2, and the iodine-131 labeled Caerin1.1 polypeptide is a Caerin1.1 polypeptide in which radioactive labeled iodine-131 is bound to the amino acid group.

[0031] The present invention also provides the use of a Caerin1.9 polypeptide or an iodine-131 labeled Caerin1.9 polypeptide in the preparation of a drug for treating prostate cancer, characterized in that the amino acid sequence of the Caerin1.9 polypeptide is GLFGVLGSIAKHVLPHVVPVIAEKL-NH2, and the iodine-131 labeled Caerin1.9 polypeptide is a Caerin1.9 polypeptide having radioactive labeled iodine-131 bonded to the amino acid group.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a new small molecule polypeptide TCMP-Y1, which is separated and identified from toad skin secretions and is a polypeptide derived from amphibians, along with Caerin1.1 and Caerin1.9. 131 I-Caerin1.1 peptide, 131 I-Caerin1.9 peptide and 131 I-TCMP-Y1 polypeptide can bind to prostate cancer cells and stably retain them, inhibiting tumor cell proliferation and tumor growth both in vivo and in vitro. 131 I-TCMP-Y1 is mainly excreted through the kidney and urinary system, thereby reducing the risk of radiation-induced liver damage. The new small molecule polypeptide TCMP-Y1 with anti-tumor activity provided by the present invention provides more options for tumor treatment. At the same time, the present invention expands the Caerin1.1 polypeptide, Caerin1.9 polypeptide, 131 I-Caerin1.1 peptide and 131 Scope of application of I-Caerin1.9 polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The effects of different concentrations of Caerin1.1 polypeptide, Caerin1.9 polypeptide, and TCMP-Y1 polypeptide on the proliferation of DU145 cells and their IC50 values. Figure 1 A, 1B, 1C: The survival rate of DU145 cells under the action of different concentrations of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide. Figure 1 D: IC of Caerin1.1 peptide, Caerin1.9 peptide and TCMP-Y1 peptide on DU145 cells 50 (ns P>0.05; **P<0.01; ****P<0.0001).

[0035] Figure 2This is a cloning diagram after different concentrations of Caerin1.1 polypeptide and Caerin1.9 polypeptide acted on DU145 cells.

[0036] Figure 3 Caerin1.1 peptide and Caerin1.9 peptide inhibit the proliferation of DU145 cells. The number of cell clones under different drug concentrations was compared with the control group (drug concentration of 0 μg / mL), and statistical analysis was performed after counting using Image-J software. (ns P>0.05; *P<0.05; **P<0.01; ****P<0.0001).

[0037] Figure 4 for 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 is a graph of gamma counts measured by paper chromatography. Figure 4 A, 4B, and 4C are 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The curve of gamma count value of I-TCMP-Y1 measured by paper chromatography showed that the labeling rate of both was greater than 90%.

[0038] Figure 5 The three labeled products were radiochemically pure under different environments. 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1, 131 I-Caerin1.1 or 131 I-Caerin1.9 or 131 The radiochemical purity (RCP) of the mixture of I-TCMP-Y1 and fetal bovine serum or normal saline stored at room temperature (25°C) and 37°C for different time periods (0h, 24h, 72h).

[0039] Figure 6 Cell uptake and elution test results. Figure 6 A shows the expression of DU145 cells at different time points (2h, 4h, 6h, 24h) and Na- 131 I. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 binding rate; Figure 6 B is the drug combined with DU145 cells and then eluted after 24 hours of incubation. At different time points (2h, 4h, 6h, 24h), Na-131 I. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 binding to DU145 cells.

[0040] Figure 7 Figure 7A, 7B, and 7C represent the results of the cell toxicity and proliferation experiment. 131 I comparison, different radioactive concentrations (2500KBq / mL, 5000KBq / mL, 10000KBq / mL, 20000KBq / mL) 131 I-Caerin1.1, 131 I-Caerin1.9, 131 Comparison of the survival rate of DU145 cells under the action of I-TCMP-Y1; Figure 7 Tables D, 7E, and 7F represent different drug concentrations (2.8 μg / mL, 5.6 μg / mL, 11.2 μg / mL, and 22.4 μg / mL) 131 I-Caerin1.1 and Caerin1.1, 131 I-Caerin1.9 and Caerin1.9, (7μg / mL, 14μg / mL, 28μg / mL, 56μg / mL) 131 Comparison of the survival rates of DU145 cells under the action of I-TCMP-Y1 and TCMP-Y1; Figure 7 G, 7H, and 7I represent different radioactive concentrations. 131 I-Caerin1.1 and 131 I-Caerin1.9, 131 I-Caerin1.1 and 131 Between I-TCMP-Y1, 131 I-Caerin1.9 and 131 Comparison of DU145 cell viability between I-TCMP-Y1; Figure 7 J, 7K, and 7L represent the comparison of DU145 cell survival rates between Caerin1.1 and Caerin1.9, between Caerin1.1 and TCMP-Y1, and between Caerin1.9 and TCMP-Y1 at different drug concentrations, respectively (ns P>0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0041] Figure 8The changes of tumor volume, tumor weight and nude mouse body weight in different treatment groups. A is the tumor volume of DU145 tumor-bearing nude mice during treatment (upper); B is the tumor volume of DU145 tumor-bearing nude mice during treatment (lower); C is the change of nude mouse body weight during treatment; D is the weight of the tumor separated during treatment. (ns P>0.05; **P<0.01; ****P<0.0001).

[0042] Fig. 9 Photos of tumors separated in different treatment groups.

[0043] Fig.10 Figure 1 is a H&E staining diagram of tumor sections in different treatment groups and a statistical diagram of the differences in the areas of cell degeneration and necrosis between groups. A is a H&E staining diagram of tumor sections in different treatment groups; B is a statistical diagram of the differences in the areas of cell degeneration and necrosis between different treatment groups. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] (1) Cell lines and cell culture

[0047] Human prostate cancer cells (DU145) were purchased from the Stem Cell Bank of the Chinese Academy of Sciences and cultured in an incubator (Thermo, USA) at 37°C and 5% carbon dioxide.

[0048] (2) Amino acid sequence composition of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide:

[0049] Caerin1.1 peptide: GLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2;

[0050] Caerin1.9 peptide: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2;

[0051] TCMP-Y1 peptide: YFKAWGWPIDDATTEHL-NH2.

[0052] Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide were all commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis and purification. The purity of the three polypeptides was determined by High Performance Liquid Chromatography (HPLC) and was >95%. The structural formulas of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide are shown below:

[0053]

[0054] Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide were dissolved in phosphate buffer solution (PBS, GIBCO, USA) to form a 1 mg / mL polypeptide solution, and stored in a -20°C refrigerator.

[0055] (3) Nude mice

[0056] 4-6 weeks old, specific pathogen-free (SPF) BALB / C male nude mice were purchased from Guangdong Medical Laboratory Animal Center (License No.: SCYK ​​(Yue) 2018-0002) and raised under SPF conditions in the SPF Laboratory Animal Center of the First Affiliated Hospital of Guangdong Pharmaceutical University (License No.: SYXK (Yue) 2017-0124), providing sterile feed, water, and an environment with 12 hours of light + 12 hours of darkness. At the end of the experiment, mice were euthanized by cervical dislocation in accordance with the "Implementation Rules for the Management of Medical Laboratory Animals" of the Ministry of Health of the People's Republic of China (No. 55).

[0057] The present invention studies and compares Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide and their iodine-131 labeled products: 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The proliferation inhibition effect of I-TCMP-Y1 on prostate cancer cell DU145 in vitro and its therapeutic value on prostate cancer in vivo are shown in the following examples.

[0058] All statistical analyses were performed using SPSS 26.0 software, and mean ± SD was used to describe the data. The t-test was used between two samples to test whether the data conformed to the normal distribution. ANOVA analysis of variance was used to compare three or more groups. GraphPad 9.0 was used to draw and present the experimental results. When P < 0.05, it was considered statistically significant.

[0059] Example 1 MTT experiment to investigate the inhibitory effects of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide on DU145 cell proliferation

[0060] In this example, the survival rate of DU145 cells was detected by MTT method, and the toxic and proliferative effects of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide on prostate cancer cell DU145 cells in vitro were studied. The specific method steps are as follows:

[0061] (1) DU145 cells with good growth status and cell density of 80% to 90% were selected for MTT experiment. DU145 cells were disinfected, rinsed, digested, counted, centrifuged, and then plated into a 96-well plate. The above DU145 cell suspension was added to each well except the outermost circle. The cell density was 5×10 3 cells / 100 μL / well; add 100 μL of sterile PBS to the outermost circle. After the plating is completed, place the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours; when the cell density is about 80%, the drug can be added;

[0062] (2) thawing the 1 mg / mL Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP-Y1 polypeptide prepared in advance at room temperature and setting aside;

[0063] (3) Set up a zero adjustment group (add only 100 μL PBS); a control group (add only 100 μL DU145 cell suspension); and three experimental groups (add 100 μL DU145 cell suspension and drugs): Caerin1.1 peptide group, Caerin1.9 peptide group, and TCMP-Y1 peptide group. Eight concentration gradients were set for the three experimental groups, namely: 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, and 40 μg / mL, with 3 replicate wells in each group, and the experiment was repeated three times;

[0064] (4) After adding drugs according to the above scheme, the 96-well plate was placed in an incubator for continued culture;

[0065] (5) After 24 h, 10 μL of MTT solution was added to each well, and the 96-well plate was then wrapped with tin foil to avoid light and placed in an incubator for further culture;

[0066] (6) After 4 h, the culture was terminated, the supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were placed on a shaker and shaken at a low speed for 10 min.

[0067] (7) Measure the OD value of each well using an ELISA reader (wavelength 570 nm). Calculate the cell survival rate = (average OD value of each experimental group - average OD value of the zero-adjustment group) / (average OD value of the control group - average OD value of the zero-adjustment group) × 100%.

[0068] (8) Using the cell viability of the three drugs obtained in the above MTT experiment, the IC of the three peptides was calculated using GraphPad Prism9.0.0 software. 50 .

[0069] The results of MTT assay are as follows Figure 1 As shown, specifically Figure 1 As shown in A, 1B, and 1C, when the concentration of Caerin1.1 peptide and Caerin1.9 peptide was 15 μg / mL, the cell survival rate was significantly reduced, which was (35.28±1.36)% and (41.86±0.91)%, respectively. When the concentration reached 20 μg / mL, the cell survival rate was only (20.32±0.38)% and (33.18±2.66)%, while when the concentration of TCMP-Y1 peptide reached 20 μg / mL, the cell survival rate was still very high, which was (100.74±2.16)%, which was significantly higher than that of Caerin1.1 peptide group and Caerin1.9 peptide group. When the peptide concentration increased to 40 μg / mL, the ability of Caerin1.1 and Caerin1.9 peptides to inhibit the proliferation of prostate cancer cells was more significant, while TCMP-Y1 peptide had no significant inhibitory effect on the proliferation of DU145 cells. Calculation of IC of Caerin1.1 peptide and Caerin1.9 peptide 50 10.87μg / mL and 14.50μg / mL respectively ( Figure 1 D). From the above results, it can be seen that when the concentration of Caerin1.1 polypeptide and Caerin1.9 polypeptide is less than 5μg / mL, the inhibitory ability of the two on DU145 cells is not significant, but as the concentration of the two polypeptides increases successively, the ability of Caerin1.1 polypeptide and Caerin1.9 polypeptide to inhibit DU145 cells gradually becomes obvious, and the higher the concentration, the lower the cell survival rate. With the increase of the concentration of toad polypeptide TCMP-Y1, the proliferation ability of DU145 cells is not significantly inhibited, so it can be inferred that TCMP-Y1 polypeptide has no ability to inhibit the proliferation of DU145 cells.

[0070] Example 2 Plate cloning experiment

[0071] (1) Select DU145 cells with good growth status and cell density of 80% to 90% for plate cloning experiment. After disinfection, rinsing, digestion, counting and centrifugation, the DU145 cells are evenly inoculated into two 6-well plates with a cell density of 800 cells / mL / well. Gently shake the 6-well plates to make the cells evenly distributed and place them in an incubator for 24 hours;

[0072] (2) After 24 hours, the cells were observed to be well attached under a microscope, and cell culture medium containing different concentrations of Caerin1.1 polypeptide and Caerin1.9 polypeptide was added to each group. The concentrations of the Caerin1.1 group were 0μg / mL, 2μg / mL, 4μg / mL, 6μg / mL, 8μg / mL and 10μg / mL; the concentrations of the Caerin1.9 group were 0μg / mL, 2.5μg / mL, 5μg / mL, 7.5μg / mL, 10μg / mL and 12.5μg / mL. After mixing evenly, the cells were placed in the incubator and cultured for 24 hours.

[0073] (3) After 24 hours, rinse and change the medium; change the medium every other day, and observe the cell status and clone number under a microscope. When the cell density of the cell clusters in the wells with a drug concentration of 0 μg / mL is about 50 / cluster, terminate the culture. The total culture period is 9 days;

[0074] (3) After terminating the culture, the culture medium was removed, the cells were washed three times with pre-cooled PBS, and 1 mL of 4% paraformaldehyde solution was added to each well for fixation for 30 min;

[0075] (4) Remove the fixative solution, rinse three times, add 0.5 mL of crystal violet staining solution to each well, and stain for 15 min; discard the crystal violet solution, rinse slowly with running water for about 15 min, and then dry naturally. Scan with a scanner or take a photo.

[0076] The results of the plate cloning experiment are as follows Figures 2-3 As shown in the figure, the plate clone formation experiment also further showed that Caerin1.1 peptide and Caerin1.9 peptide can inhibit the proliferation of prostate cancer cell DU145. With the increase of peptide concentration, the number of cells gradually decreased and the proliferation ability decreased. Compared with the control group (concentration of 0μg / mL), the difference in the number of cell clones was statistically significant (P<0.05), which was consistent with the results of MTT experiment.

[0077] Based on the experimental results of Examples 1 and 2, it can be seen that Caerin1.1 polypeptide and Caerin1.9 polypeptide have a significant inhibitory effect on the proliferation of prostate cancer cells (DU145 cells), and the inhibitory effect is concentration-dependent; while TCMP-Y1 polypeptide has no inhibitory effect on the proliferation of prostate cancer cells DU145, indicating that it has no anti-tumor activity. The plate clone formation experiment further verified that Caerin1.1 polypeptide and Caerin1.9 polypeptide can significantly inhibit the growth of DU145 tumor cells, and is concentration-dependent.

[0078] Example 3 131 Preparation and quality identification of I-labeled Caerin1.1, Caerin1.9 and TCMP-Y1 polypeptides

[0079] Preliminary experiments have shown that 100 μL of (370 MBq / mL, 10 mCi / mL, i.e., a total of 1 mCi) Na 131 The labeling rate was the highest when I solution was labeled with 40 μL (1 mg / mL) Caerin1.1 or Caerin 1.9 peptide, so the subsequent labeling was prepared using this ratio. 131 I-Caerin1.1 and 131 I-Caerin 1.9. In addition, according to preliminary experiments, 131 When TCMP-Y1 peptide is labeled with I, 100 μL (370 MBq / mL, 10 mCi / mL, i.e., a total amount of 1 mCi) Na 131 The labeling rate is highest when I solution is labeled with 100 μL (1 mg / mL) TCMP-Y1 peptide. The specific operation steps are as follows:

[0080] (1) Take 40 μL (1 mg / mL) of Caerin1.1 polypeptide, 40 μL (1 mg / mL) of Caerin1.9 polypeptide and 100 μL (1 mg / mL) of TCMP-Y1 polypeptide and place them in three 1.5 mL sterile EP tubes for later use;

[0081] (2) Dissolve 50 mg of chloramine-T trihydrate (McLean, China) in 50 mL of PBS to a concentration of 1 mg / mL and store in the dark;

[0082] (3) Customized three Na 131 I solution, the volume was 100 μL, and the radioactive concentration was 370 MBq / mL (10 mCi / mL) measured by activity meter; 100 μL of Na 131 I solution, followed by the addition of 100 μL of 1 mg / mL chloramine-T solution;

[0083] (4) Mix and oscillate for 10 min using a vortex mixer at room temperature. 131 I labeling experiment of Caerin1.1, Caerin1.9, and TCMP-Y1.

[0084] (4) The radioactivity of the samples was evaluated by thin layer paper chromatography using physiological saline (NS, Yangzhou Zhongbao Pharmaceutical, China) as the developing solvent and a γ-counter (Zhongjia Optoelectronics, China) to measure the radioactivity. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Labeling rate of I-TCMP-Y1 The γ counting curve was drawn using GraphPad Prism 9.0.0 software, and the labeling rate was obtained by calculating the area under the curve.

[0085] Marking rate = 131 I-Caerin1.1 or 131 I-Caerin1.9 or 131 The integrated radioactive peak area of ​​I-TCMP-Y1 / the integrated radioactive peak area of ​​the whole body × 100%.

[0086] Rf value = 131 I-Caerin1.1 or 131 I-Caerin1.9 or 131 I- Distance moved by TCMP-Y1 / Distance moved by the developing agent front.

[0087] The results are as follows Figure 4 As shown, the calculated 131 I-Caerin1.1, 131 I-Caerin1.9, 131 The radioactive labeling rates of I-TCMP-Y1 were (96.86%±0.54)%, (95.39%±0.50)%, and (94.92%±0.14)%, respectively, and all of them were around 95%.

[0088] Example 4 131 I-Caerin 1.1, 131 I-Caerin1.9 and 131 Determination of lipid-water partition coefficient of I-TCMP-Y1

[0089] This embodiment uses 131 I-Caerin 1.1, 131 I-Caerin 1.9 and 131 Determination of the lipid-water partition coefficient of I-TCMP-Y1 to infer the excretion pathways of the three radiolabeled products in the body. The operation is as follows:

[0090] (1) Prepare three EP tubes, each filled with 500 μL of n-octanol (lipid phase) and 500 μL of saline (water phase);

[0091] (2) Prepared according to the above labeling method 131 I-Caerin1.1, 131 I-Caerin1.9 or 131 I-TCMP-Y1, respectively, pipette 50 μL into EP tubes, seal and mix;

[0092] (3) Place the EP tube in a centrifuge, balance it, and centrifuge it at 4000 rpm / min for 5 min. After centrifugation, you can see that the solution is clearly separated into layers, with the upper layer being the lipid phase and the lower layer being the water phase.

[0093] (4) 100 μL of liquid was taken from the upper lipid phase and the lower aqueous phase and placed in two glass counting tubes respectively, and the gamma counts were measured using a gamma radioimmunoassay counter;

[0094] (5) Calculate the lipid-water partition coefficient (log P), log P = log [(lipid phase γ counts - background γ counts) / (aqueous phase γ counts - background γ counts)].

[0095] Table 1 131 CPM counts and lipid-water partition coefficient of I-Caerin1.1 in lipid and aqueous phases

[0096]

[0097] Table 2 131 CPM counts and lipid-water partition coefficient of I-Caerin1.9 in lipid and aqueous phases

[0098]

[0099] Table 3 131 CPM count and lipid-water partition coefficient of I-TCMP-Y1 in lipid and water phases

[0100]

[0101] 131 I-Caerin 1.1, 131 I-Caerin1.9 and 131 The lipid-water partition coefficients of I-TCMP-Y1 are shown in Tables 1, 2 and 3, respectively. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131The lipid-water partition coefficients Log P of I-TCMP-Y1 were 0.137±0.048 (n=4), 0.098±0.013 (n=4) and -0.478±0.030 (n=4), respectively. 131 I-Caerin1.1 and 131 I-Caerin1.9 is weakly lipid soluble, 131 I-TCMP-Y1 is weakly water-soluble.

[0102] Example 5 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Determination of the stability of I-TCMP-Y1

[0103] In this embodiment, 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 was placed in fetal bovine serum or saline at 25°C or 37°C, and its radiochemical purity (RCP) was measured at different time points to evaluate its in vitro stability. The experiment was repeated three times, and the operation was as follows:

[0104] (1) Place 100 μL of 131 I-Caerin1.1, 131 I-Caerin1.9 or 131 I-TCMP-Y1, and stored in a sealed constant temperature water tank at 37°C and room temperature (25°C);

[0105] (2) Take 20 μL of each labeled solution and place it in 80 μL of fetal bovine serum or 80 μL of normal saline, mix well using a vortex mixer, and store at room temperature at 25°C;

[0106] (3) Take 20 μL of each labeled solution and place it in 80 μL of fetal bovine serum or 80 μL of normal saline, mix well using a vortex mixer, and store in a constant temperature water tank at 37°C;

[0107] (4) The radiochemical purity of each mixture at different time periods (0 h, 24 h and 72 h) was detected by paper chromatography, and the experiment was repeated three times.

[0108] The results are as follows Figure 5 As shown in the figure, the three labeled products all had high radiochemical purity (RCP) after being placed in fetal bovine serum (FBS) or normal saline (NS) at room temperature (25°C) or 37°C for different periods of time. Figure 5 A. 5D, 5G,131 I-Caerin1.1, 131 I-Caerin1.1 and fetal bovine serum mixture 131 After the mixture of I-Caerin1.1 and saline was stored at 25°C for 72 hours, the radiochemical purity was (84.62±1.78)%, (85.19±1.49)%, and (84.81±0.79)%, respectively. After the mixture was placed in a constant temperature water tank at 37°C for 72 hours, the RCP was (84.41±0.79)%, (85.36±1.08)%, and (85.12±1.77)%, respectively. Figure 5 As shown in B, 5E, and 5H, 131 I-Caerin1.9, 131 I-Caerin1.9 and fetal bovine serum mixture 131 After the mixture of I-Caerin1.9 and saline was stored at 25℃ for 72h, the radiochemical purity was (85.99±2.23)%, (85.87±1.14)%, and (85.82±2.17)% respectively; after being placed in a 37℃ constant temperature water tank for 72h, the RCPs were (86.42±2.46)%, (87.54±0.79)%, and (87.26±1.10)% respectively. Figure 5 C, 5F, 5I, simple 131 I-TCMP-Y1 sample, 131 I-TCMP-Y1 and fetal bovine serum mixture 131 After the mixture of I-TCMP-Y1 and physiological saline was stored at 25°C for 72 hours, the radiochemical purity was (87.70±0.60)%, (87.48±0.82)%, and (87.78±1.19)%, respectively; after being placed in a constant temperature water tank at 37°C for 72 hours, the radiochemical purity of the three were (88.09±1.05)%, (87.71±0.74)%, and (85.94±1.43)%, respectively.

[0109] The above experimental results show that 131 I-Caerin1.1, 131 I-Caerin1.9 or 131 I-TCMP-Y1, the three labeled products still have high in vitro stability after being stored in different temperatures (25℃, 37℃) and different solutions (fetal bovine serum or saline) for 72 hours.

[0110] Based on the experimental results of Examples 3 to 5, it can be seen that the chloramine-T direct labeling method 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The labeling rate of I-TCMP-Y1 is about 95%.131 I-Caerin1.1 and 131 I-Caerin1.9 is weakly lipid-soluble and may be excreted through the liver; 131 I-TCMP-Y1 is weakly water soluble and may be excreted through the kidneys. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has good in vitro stability in fetal bovine serum or normal saline at different temperatures (25℃, 37℃).

[0111] Example 6 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Cellular uptake experiment of I-TCMP-Y1

[0112] In this example, DU145 cells were tested at different time points. 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1 and Na 131 The uptake of I by DU145 cells was evaluated 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1 and Na 131 The binding capacity of I is as follows:

[0113] (1) DU145 cells in the logarithmic growth phase were selected and digested into a cell suspension (see 2.3.4 Cell Digestion for specific steps), then counted and resuspended to 1×10 5 Pieces / mL;

[0114] (2) The above cell suspension was evenly inoculated in 8 24-well plates, with the number of cells added being 5×10 4 pcs / 500 μL / well, and continue to culture in the incubator for 24 h;

[0115] (3) After the cells adhered to the wall, the supernatant was discarded, and the cells were rinsed twice. 0.5 mL of serum-free pure culture medium was added to each well and divided into 4 experimental groups, with 3 replicate wells and 1 positive control well in each group. The drug concentrations were as follows:

[0116] A: 131 I-Caerin1.1 group: 259KBq / 2μL / well;

[0117] B: 131 I-Caerin1.9 group: 259KBq / 2μL / well;

[0118] C: 131 I-TCMP-Y1 group: 259KBq / 2μL / well;

[0119] D:Na 131 Group I: 259KBq / 2μL / well;

[0120] (4) After adding drugs according to the above scheme, the 24-well plate was placed in an incubator and cultured for 2, 4, 6, and 24 hours respectively; after the culture was completed, the supernatant was aspirated and washed with fresh pre-cooled PBS, and repeated twice (the supernatant and washing solution of the positive control wells were collected in the corresponding test tubes), and all wells were digested with 200 μL of trypsin, and washed with 400 μL of fresh pre-cooled PBS each time, repeated 3 times and the washing solution was collected;

[0121] (5) Measure the radioactivity count in each test tube and calculate the cell binding rate at different time points.

[0122] Cell binding rate = (average γ counts of three replicate wells in each group / γ counts of the corresponding positive control wells in each group) × 100%.

[0123] The results of the cell uptake experiment are as follows Figure 6 As shown in A, in the cell uptake experiment, DU145 cells were 131 I-Caerin1.1, 131 I-Caerin1.9, 131 The binding rate of I-TCMP-Y1 increased with the extension of time, and reached the highest at 24h, with the binding rates of (19.60±0.81)%, (18.65±0.53)%, and (9.00±1.45)% respectively; and 131 I-Caerin1.1, 131 I-Caerin1.9 grew 131 I-TCMP-Y1 is faster; however, DU145 cells have little ability to take up Na 131 The binding rate of I in 24 hours was only (0.57±0.03)%.

[0124] Example 7 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Cell elution experiment of I-TCMP-Y1

[0125] (1) Select logarithmically growing DU145 cells, digest, count, centrifuge, and resuspend to 1×10 5 Pieces / mL;

[0126] (2) Inoculate the above cell suspension in 8 24-well plates, with an inoculation volume of 500 μL per well, and continue culturing in an incubator for 24 h;

[0127] (3) After the cells adhered to the wall, the supernatant was aspirated, the cells were rinsed twice, 0.5 mL of serum-free medium was added to each well, and four experimental groups were divided, with 3 replicate wells and 1 positive control well in each group; the drug concentrations were as follows:

[0128] A: 131 I-Caerin1.1 group: 259KBq / 2μL / well;

[0129] B: 131 I-Caerin1.9 group: 259KBq / 2μL / well;

[0130] C: 131 I-TCMP-Y1 group: 259KBq / 2μL / well;

[0131] D:Na 131 Group I: 259KBq / 2μL / well;

[0132] (4) Continue culturing in the incubator for 24 hours; after 24 hours, discard the supernatant, rinse twice, add 0.5 mL of serum-free medium to each well, and culture in the incubator for 2 hours, 4 hours, 6 hours, and 24 hours, respectively; then take out the 24-well plate, aspirate the supernatant, wash with pre-cooled PBS, repeat twice (collect the supernatant and rinse solution of the positive control well in the corresponding test tube), add 200 μL of trypsin to digest the cells in each well, wash with fresh pre-cooled PBS, repeat 3 times and collect the rinse solution;

[0133] (5) Determine the γ count of each test tube and calculate the cell binding rate of the four groups at each time point. Cell binding rate = (average γ count of three replicate wells in each group / γ count of the corresponding positive control well in each group) × 100%.

[0134] The results of the cell elution experiment are as follows Figure 6 B indicates that when 131 I-Caerin1.1, 131 I-Caerin1.9, 131 I-TCMP-Y1 was combined with DU145 cells and incubated for 24 hours before elution. The binding rates of the three drugs with DU145 cells were all high ( 131 The binding rate of I-TCMP-Y1 to DU145 cells was slightly lower than that of the other two groups of drugs, and tended to be flat at 24h. After elution and then cultured for 24h, the binding rates were (61.64±1.07)%, (46.68±1.64)%, and (66.65±0.80)%, respectively. 131 The binding rate of I was only (3.03±0.21)%.

[0135] The above cell uptake and elution experiments showed that the three radiolabeled products could be taken up by DU145 cells.

[0136] Example 8 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 Cytotoxicity and proliferation assay of I-TCMP-Y1

[0137] The CCK-8 method was used to verify that DU145 cells were expressed in Caerin1.1 peptide, Caerin1.9 peptide, TCMP-Y1 peptide, and Na 131 I. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The differences in cell survival rates under the action of I-TCMP-Y1 were compared to compare the differences in the inhibition of DU145 proliferation by different drugs. The specific experimental method steps are as follows:

[0138] (1) DU145 cells in logarithmic growth phase with a cell density of 80% to 90% were selected, digested, centrifuged, counted, and resuspended to 5.0×10 4 100 μL of cell suspension was added to each well of the 96-well plate using a spray gun, and the cell density was 5×10 3 100 μL of sterile PBS was added to the edge wells of the 96-well plate. After all the cells were added, the plate was gently shaken to mix the cells evenly and then moved to a 37°C, 5% CO2 incubator for 24 hours.

[0139] (2) After 24 hours, when the cells have grown to 80% of the wells, the drug is added. The drug concentrations for each group are as follows:

[0140] A: 131 I-Caerin1.1 group: 2500KBq / mL, 5000KBq / mL, 10000KBq / mL, 20000KBq / mL; 131 I-Caerin1.9 group, 131 I-TCMP-Y1 group and Na 131 Group I 131 I-Caerin1.1 group;

[0141] B: According to the amount of group A, the corresponding Caerin1.1 polypeptide group and Caerin1.9 polypeptide group were 2.8μg / mL, 5.6μg / mL, 11.2μg / mL, and 22.4μg / mL, respectively, and the corresponding TCMP-Y1 polypeptide group were 7μg / mL, 14μg / mL, 28μg / mL, and 56μg / mL, respectively; 3 parallel wells were set for each group, and three wells without drug addition were used as the control group;

[0142] (3) After continuing the culture for 24 hours, 10 μL of CCK-8 reagent was added to each well and the cells were cultured in the dark for 4 hours. The OD value of each well was detected using a full-wavelength microplate reader (wavelength 450 nm) to calculate the survival rate of each group of cells.

[0143] Cell survival rate = (average OD value of each experimental group - average OD value of the zero-adjustment group) / (average OD value of the control group - average OD value of the zero-adjustment group) × 100%.

[0144] The experimental results are as follows Figure 7 As shown, Figure 7 A, 7B, 7C, when 131 I-Caerin1.1, 131 When the radioactivity concentration of I-Caerin1.9 reaches 20000KBq / mL, 131 I-Caerin1.1 group, 131 The survival rates of DU145 cells in the I-Caerin1.9 group were (7.18±0.93)% and (19.58±1.80)% respectively; 131 The cell survival rate of I-TCMP-Y1 group was still as high as (39.17±0.83)%. Figure 7 D, 7E, and 7F are compared with the simple peptide group with the same peptide concentration. 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131 The cell survival rates of the I-TCMP-Y1 group were lower, and the differences were statistically significant (P < 0.05). 131 I only exerted an inhibitory effect on the cell proliferation of prostate cancer cell DU145, and the cell survival rate was (70.09±3.39)%. Figure 7 G, 7H, 7I, at higher concentrations, 131 I-Caerin1.1 and 131 I-Caerin1.9, 131 I-Caerin1.1 and 131 Between I-TCMP-Y1, 131I-Caerin1.9 and 131 The comparison of the toxic effects of I-TCMP-Y1 on DU145 cells showed statistical differences (P < 0.05). For example, at higher concentrations, Caerin1.1 peptide and Caerin1.9 peptide, Caerin1.1 peptide and TCMP-Y1 peptide, Caerin1.9 peptide and TCMP-Y1 peptide had statistical differences in their inhibitory effects on DU145 cell proliferation (P < 0.05), which is consistent with the results of the previous MTT experiment. From the above results, it can be seen that the CCK-8 experiment verified 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 can inhibit the proliferation of DU145 cells. With the increase of radioactive concentration, the cytotoxic effects of the three radioactive drugs gradually increased and the survival rate of DU145 cells gradually decreased.

[0145] Example 9 Establishment of DU145 cell tumor-bearing nude mouse model

[0146] (1) Select DU145 cells in the logarithmic growth phase with a cell density of 80% to 90%. After digestion, centrifugation, and counting, resuspend the cells in pre-cooled sterile PBS and adjust the cell concentration to 1×10 7 / mL, transfer the cell suspension to a 5mL EP tube and place it in an ice box;

[0147] (2) After the local skin of the right upper arm of the nude mouse was disinfected with alcohol disinfectant, 100 μL of the above DU145 cell suspension was drawn into the skin with a syringe. The number of cells was 1×10 6 The inoculation was slowly carried out into the axillary skin, and a raised transparent blister was visible under the right axillary of the nude mouse.

[0148] (3) After about two days, the blisters are absorbed and you can see the formation of rice-like growths on the right armpit skin. Regularly observe the condition of the nude mice and the growth of the tumor, and replace fresh feed and water; measure the tumor size with an electronic vernier caliper every two days. When the tumor size is close to 3-4 mm, it means that the prostate cancer nude mouse model has been successfully established and subsequent experiments can be carried out. Tumor volume = width × width × length / 2.

[0149] The results are as follows Figure 8 As shown by Figure 8 A and 8B show that before euthanasia, the PBS group, Caerin1.1 group, Caerin1.9 group, TCMP-Y1 group, and Na 131 Group I, 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and131 The tumor volume of the I-TCMP-Y1 group was as follows: (344.49±57.96) mm 3 、(373.87±54.99)mm 3 、(339.17±70.21)mm 3 、(333.56±32.32)mm 3 、(330.86±60.83)mm 3 、(33.96±5.56)mm 3 、(37.70±1.72)mm 3 and (69.76±5.20)mm 3 Compared with before treatment, the tumor volume of each group increased, but the tumor volume of the three groups labeled with iodine-131 peptide increased significantly compared with the PBS group, 131 At the beginning of treatment, there was no statistical difference in the tumor volume of nude mice in each group (P>0.05). Figure 8 C, from the end of treatment to euthanasia, the weights of nude mice in each group were: (22.57±0.89)g, (22.83±1.43)g, (22.98±0.71)g, (24.15±0.63)g, (25.01±0.21)g, (23.55±2.03)g, (24.34±2.01)g and (23.11±0.55)g. It can be seen that the weights of nude mice in each group increased compared with those before treatment. Figure 8 D, After the nude mice were killed, they were dissected, the tumor tissues were separated and weighed. The weights of the tumors in each group were as follows: (0.190±0.004)g, (0.146±0.008)g, (0.150±0.013)g, (0.146±0.015)g, (0.158±0.021)g, (0.023±0.008)g, (0.046±0.006)g and (0.069±0.008)g. 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131 The tumor size of the I-TCMP-Y1 group was significantly smaller than that of the other groups, proving that 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The I-TCMP-Y1 group also has the effect of inhibiting the growth of prostate cancer in vivo.

[0150] Example 10 131 I-Caerin1.1, 131 I-Caerin1.9 and131 In vivo therapeutic experiments with I-TCMP-Y1

[0151] (1) Before the in vivo experiment, in order to block the thyroid gland and reduce the thyroid uptake of nude mice 131 I, nude mice used in the experiment were fed with 0.1% potassium iodide 3 days in advance;

[0152] (2) The successfully modeled DU145 tumor-bearing nude mice were randomly divided into 8 groups, with 4 mice in each group, namely, Caerin1.1 polypeptide group, Caerin1.9 polypeptide group, TCMP-Y1 polypeptide group, PBS group (control group), 131 I-Caerin 1.1 group, 131 I-Caerin1.9 group, 131 I-TCMP-Y1 group, Na- 131 In group I, the drug volume for each nude mouse was 100 μL per treatment. The Caerin1.1 peptide group and the Caerin1.9 peptide group were given 100 μL PBS solution containing 8 μg Caerin1.1 peptide or 8 μg Caerin1.9 peptide; the TCMP-Y1 peptide group was given 100 μL PBS solution containing 20 μg TCMP-Y1 peptide; the PBS group (control group) was given 100 μL sterile PBS solution; Na 131 Group I was given sterile Na 131 I solution; 131 I-Caerin1.1 group and 131 The I-Caerin1.9 group was given 8 μg Caerin1.1 polypeptide or 8 μg Caerin1.9 polypeptide and 200 μCi Na 131 A mixture of I; 131 The I-TCMP-Y1 group was given a solution containing 20 μg TCMP-Y1 peptide and 200 μCi Na 131 A mixture of I;

[0153] (3) The drug was injected intratumorally once every 2 days for a total of 3 times. Before each injection, the length and width of the nude mouse tumor were measured with an electronic vernier caliper and the volume was calculated. The nude mouse body weight was weighed with an electronic balance. On the 5th day after the last treatment, the nude mouse was killed by cervical dislocation, and the tumor was dissected and weighed.

[0154] The experimental results are as follows Fig. 9 As shown in Figure 2, after nude mice were euthanized, tumor tissue specimens isolated from different treatment groups were obtained. 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131The tumor volume of the I-TCMP-Y1 group was smaller overall, indicating 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has anti-prostate cancer activity.

[0155] The results of H&E staining of tumor sections in different treatment groups and the statistical diagram of the differences in cell degeneration and necrosis areas in different treatment groups are shown in the figure below. Fig.10 As shown in the figure, the tumor cells in the PBS group were closely arranged, with large and darkly stained nuclei, which was called proliferation-like changes, and a small number of cells were observed to be degenerated and necrotic. A small number of cells were observed to be degenerated and necrotic in the Caerin1.1 group, Caerin1.9 group, and TCMP-Y1 group, and there was no significant difference compared with the PBS control group (P>0.05). 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 tumor cells in all three groups showed more degeneration and necrosis, showing homogeneous red staining, and the cell structure was also destroyed ( Fig.10 A). 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131 I-TCMP-Y1 group and PBS group 131 Compared with group I, the area of ​​tumor necrosis was significantly different (P < 0.05). 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131 The tumor necrosis area in the I-TCMP-Y1 group was significantly different from that in the simple peptide group (P < 0.05). Fig.10 B) This result shows 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 can inhibit and kill tumor cells and suppress tumor proliferation in vivo.

[0156] Based on the experimental results of Examples 6 to 10, 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 can be taken up by prostate cancer (DU145) cells, and 131 I-Caerin1.1, 131 I-Caerin1.9 can be stably retained in DU145 cells. 131 The ability of I-TCMP-Y1 to remain in DU145 cells is131 I-Caerin1.1, 131 I-Caerin1.9 is weak; DU145 cells hardly take up 131 I. 131 I-Caerin1.1, 131 I-Caerin1.9 or 131 I-TCMP-Y1 has obvious proliferation inhibition and killing effects on DU145 cells; and compared with Caerin1.1 peptide and Caerin1.9 peptide at the same concentration, 131 I-Caerin1.1, 131 I-Caerin1.9 had a stronger inhibitory effect, while TCMP-Y1 had no significant inhibitory effect on DU145 cells. In the in vivo treatment experiment, compared with the control group, the simple peptide group, and the simple iodine-131 group, the tumor volume and tumor weight were observed. 131 I-Caerin 1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has a significant inhibitory effect on the growth of prostate cancer. Before the nude mice were killed, the weight of each group of nude mice increased compared with before treatment, which may indicate that 131 I-Caerin 1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has good biosafety.

[0157] The results of the MTT experiment of the present invention show that Caerin1.1 polypeptide and Caerin1.9 polypeptide can inhibit the proliferation of prostate cancer cells (DU145) in vitro, and show concentration dependence. The IC50 of Caerin1.1 and Caerin1.9 are 10.87 μg / mL and 14.50 μg / mL respectively, while TCMP-Y1 polypeptide has no obvious inhibitory effect on the proliferation of DU145. The results of the plate cloning experiment are similar to those of MTT, and both can prove that with the increase of the concentration of Caerin1.1 polypeptide and Caerin1.9 polypeptide, the drug has a more significant effect on the proliferation toxicity of DU145 cells.

[0158] The present invention adopts the chloramine-T method to prepare three 131 I-labeled product, i.e. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1, and the labeling rates of the three are all around 95%. 131 I-Caerin1.1, 131 I-Caerin1.9 and131 I-TCMP-Y1 can maintain high stability in both NS and FBS at room temperature (25°C) or human body temperature (37°C). 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The lipid-water partition coefficient of I-TCMP-Y1 is known in the present invention. 131 I-Caerin1.1, 131 The LogP values ​​of I-Caerin1.9 were both > 0, indicating that the two drugs were weakly lipid soluble. 131 The LogP of I-TCMP-Y1 is less than 0, indicating 131 I-TCMP-Y1 is weakly water soluble, which proves 131 I-Caerin1.1, 131 I-Caerin1.9 may be mainly excreted through the liver; 131 I-TCMP-Y1 may be mainly excreted from the body through the urinary system, which can reduce the risk of radiation-induced liver injury. In the cell uptake experiment and cell elution experiment, as the drug action time increases, 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 The binding rate of I-TCMP-Y1 in prostate cancer cell DU145 gradually increased, and the binding ability gradually improved. 131 I-TCMP-Y1 is weaker than the first two, but both are higher than Na 131 Group I, which indicates 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 can be taken up and stably retained by prostate cancer cells (DU145). 131 I, the reason may be related to the fact that prostate cancer cells do not express sodium iodide transporter (NIS). In the cell elution experiment, elution was performed after 2h, 4h, 6h and 24h. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has a high retention capacity in prostate cancer cells (DU145), and 131 I-Caerin1.1, 131 I-Caerin1.9 131 The binding force of I-TCMP-Y1 is stronger.

[0159] The results of CCK8 experiments confirmed that 131I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has a significant proliferative toxicity to prostate cancer cells (DU145) and is more effective than simple Na 131 I, Caerin1.1 polypeptide, and Caerin1.9 polypeptide have stronger effects, while the simple TCMP-Y1 polypeptide has no obvious toxic effect on DU145 cells. This may be because Caerin1.1 polypeptide, Caerin1.9 polypeptide, and TCMP-Y1 polypeptide can 131 This is consistent with the results of previous cell uptake experiments and cell elution experiments.

[0160] In the in vivo experiment, the present invention selected intratumoral injection of drugs as a treatment method, because the advantage of intratumoral administration is that it can avoid the problems caused by systemic administration, such as poor tumor penetration, rapid drug clearance and damage to healthy tissues. 131 Compared with group I, 131 I-Caerin1.1 group, 131 I-Caerin1.9 group and 131 The growth rate of tumor volume and tumor weight in the I-TCMP-Y1 group were lower, and the difference was statistically significant (P < 0.05); however, there was no statistical difference in the growth rate of tumor volume among the three groups of iodine-labeled polypeptides (P > 0.05); in terms of tumor weight, 131 I-Caerin1.1 group and 131 Between I-Caerin1.9 groups, 131 I-Caerin1.9 group and 131 There was no statistical difference between the I-TCMP-Y1 groups (P>0.05), while 131 I-Caerin1.1 group and 131 There was a statistically significant difference between the I-TCMP-Y1 groups (P<0.05).

[0161] Through cytotoxic proliferation experiments and in vivo experiments, the present invention found that compared with Caerin1.1, Caerin1.9, and TCMP-Y1, 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 has a stronger tumor inhibitory effect on prostate cancer. Therefore, the present invention speculates that the three polypeptides all carry 131 I The ability to enter tumor cells. 131I-Caerin1.1 and 131 I-Caerin1.9 has both the biological radiation effect of radionuclides and the anti-cancer effect of small molecule peptides, so its biological effect is better than that of Na 131 I, Caerin1.1 and Caerin1.9; According to the previous MTT experiment, the simple TCMP-Y1 polypeptide has no obvious inhibitory effect on the proliferation of DU145 cells. 131 The tumor suppressor effect of I-TCMP-Y1 may be due to 131 I radiation biological effects.

[0162] In summary, the experimental results of the present invention show that the simple TCMP-Y1 polypeptide has no obvious proliferation inhibitory effect on prostate cancer cell DU145; while the simple Caerin1.1 and Caerin1.9 have proliferation inhibitory effect on prostate cancer cell DU145. 131 I-Caerin1.1, 131 I-Caerin1.9 and 131 I-TCMP-Y1 can be taken up and stably retained by prostate cancer cell DU145, and has a more obvious effect of inhibiting tumor growth. 131 I-TCMP-Y1 is mainly excreted through the kidney and urinary system, thus reducing the risk of radiation-induced liver injury. This indicates that TCMP-Y1 is a small molecule peptide that is good at introducing radionuclides into the body, improving drug safety and tumor targeting, and can be used as a potential drug for tumor intra-irradiation therapy.

Claims

1. An iodine-131 labeled small molecule polypeptide TCMP-Y1, characterized in that: The amino acid sequence of the small molecule polypeptide TCMP-Y1 is YFKAWGWPIDDATTEHL-NH2, and radioactive labeled iodine-131 is bound to the amino acid group thereof.

2. The method for preparing the iodine-131 labeled small molecule polypeptide TCMP-Y1 according to claim 1, characterized in that: In order to use the chloramine-T method, the small molecule polypeptide TCMP-Y1 solution, Na- 131 I solution and chloramine-T solution were shaken and mixed at room temperature, and then separated by chromatography. 131 The small molecule polypeptide TCMP-Y1 labeled with I is obtained.

3. The preparation method according to claim 2, characterized in that: The TCMP-Y1 and Na- 131 The ratio of I is 40μg:1mCi.

4. Use of the iodine-131 labeled small molecule polypeptide TCMP-Y1 according to claim 1 in the preparation of a drug for treating prostate cancer.

5. A tumor treatment drug, characterized in that: Contains the iodine-131 labeled small molecule polypeptide TCMP-Y1 as claimed in claim 1.

6. The drug according to claim 5, characterized in that: Pharmaceutically acceptable excipients are also included.

7. Use of Caerin1.1 polypeptide or Caerin1.1 polypeptide labeled with iodine-131 in the preparation of a drug for treating prostate cancer, characterized in that: The amino acid sequence of the Caerin1.1 polypeptide is GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2, and the Caerin1.1 polypeptide labeled with iodine-131 is a Caerin1.1 polypeptide in which radioactive labeled iodine-131 is combined with the amino acid group of the polypeptide.

8. Use of Caerin1.9 polypeptide or Caerin1.9 polypeptide labeled with iodine-131 in the preparation of a drug for treating prostate cancer, characterized in that: The amino acid sequence of the Caerin1.9 polypeptide is GLFGVLGSIAKHVLPHVVPVIAEKL-NH2, and the Caerin1.9 polypeptide labeled with iodine-131 is a Caerin1.9 polypeptide in which radioactive labeled iodine-131 is combined with the amino acid group of the Caerin1.9 polypeptide.

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