Iodine-131-labeled small-molecule polypeptide TFMP-y2, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are an iodine-131-labeled small-molecule polypeptide TFMP-Y2, and a preparation method therefor and the use thereof. In the present invention, on the basis of the Caerin 1.9 polypeptide and by performing modification to enhance the water solubility of the polypeptide and reduce the inhibitory effect of the polypeptide on the proliferation of normal cells, a new small-molecule polypeptide TFMP-Y2 is obtained. The TFMP-Y2 polypeptide alone has no significant inhibitory effect on the proliferation of normal cells and HGC-27. However, 131I-TFMP-Y2 can be stably retained in gastric carcinoma cells, and has a significant anti-tumor effect. Moreover, the water-soluble 131I-TFMP-Y2 is mainly excreted by the renal urinary system, such that the risk of radioactive liver damage caused by 131I-Caserin 1.9 can be reduced. Therefore, TFMP-Y2 is a good small-molecule polypeptide for introducing a nuclide into the body, which improves the drug safety and the tumor targeting property, and can be used as a potential drug for internal radiation therapy of tumors.

Description

An iodine-131 labeled small molecule polypeptide TFMP-Y2 and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to an iodine-131 labeled small molecule polypeptide TFMP-Y2, and a preparation method and application thereof. Background Art

[0002] Gastric cancer (GC) is the fifth most common malignant tumor worldwide and ranks third in mortality. Due to subtle early-stage symptoms and low rates of routine screening, most patients are diagnosed at an advanced stage. Although treatment options include surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapies, efficacy remains suboptimal, resulting in a poor prognosis, with a five-year survival rate of less than 30%. Therefore, the exploration and development of novel, effective treatments to improve survival rates is becoming an inevitable trend.

[0003] Internal radiation therapy of radionuclide (IRT) refers to the introduction of radioactive labeled substances into the body through various means, using the α, β or Auger electrons emitted by the radionuclide to generate radiation, transporting the radionuclide into the body through catheter insertion, interstitial injection, particle implantation and intravenous injection of tropism drugs, etc., and using the biological effects of the ionizing radiation emitted by the nuclide to kill cancer cells or cause genetic changes leading to cancer cell death, while protecting normal tissues. In recent years, small molecule peptides and their analogs have become an international research hotspot due to their advantages such as strong tissue penetration, rapid blood clearance, low antigenicity, easy structural modification and good stability. Studies have already radioactively labeled radionuclides with a variety of small molecule peptides, which provides an effective and promising treatment method for cancer treatment. For example ( 177 Lu-DOTA-TATE) is one of the radiopharmaceuticals approved by the FDA and EMA for peptide receptor radionuclide therapy.

[0004] Caerin1.9 (GLFGVLGSIAKHVLPHVVPVIAEKL-NH2) peptide is a host defense peptide isolated and identified from the skin secretions of Australian tree frogs - one of the caerin series peptides. The inventor's team's previous research has shown that Caerin1.9 peptide inhibits and kills a variety of tumor cells including lung cancer, melanoma, cervical cancer and breast cancer, and 131 I-Caerin1.9 peptide has a stronger tumor inhibitory effect than simple Caerin1.9 peptide, but it has not yet been seen 131There are reports on the use of I-labeled caerin1.9 in the treatment of gastric cancer. In addition, high concentrations of Caerin1.9 inhibit the proliferation of normal thyroid cells, that is, Caerin1.9 has an inhibitory effect on normal cells during treatment, and 131 I-Caerin1.9, as a fat-soluble small molecule, will be excreted through the liver and gallbladder. On the one hand, it will affect the application of drugs in hepatobiliary tumors. On the other hand, a large amount of radioactive drugs will accumulate in the liver in a short period of time. When the radiation dose exceeds a certain level, it may cause radiation damage to the liver. Therefore, its safety and metabolic pathways limit its clinical application. Summary of the Invention

[0005] 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 TFMP-Y2.

[0006] The second object of the present invention is to provide an iodine-131 labeled small molecule polypeptide TFMP-Y2.

[0007] The third object of the present invention is to provide a method for preparing the iodine-131 labeled small molecule polypeptide TFMP-Y2.

[0008] The fourth object of the present invention is to provide the use of the iodine-131 labeled small molecule polypeptide TFMP-Y2 in the preparation of drugs for treating tumors.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] This study uses the Caerin 1.9 polypeptide as a basis and modifies it to enhance its water solubility and reduce its inhibitory effect on normal cell proliferation, resulting in a new small molecule polypeptide, TFMP-Y2. In vitro and in vivo experiments were conducted to investigate the polarity of Caerin 1.9 and TFMP-Y2, and to compare Caerin 1.9 with the modified polypeptide TFMP-Y2 and its iodine-131 labeled product: 131 I-Caerin1.9 peptide and 131 The therapeutic effect of I-TFMP-Y2 peptide on gastric cancer.

[0011] Specifically: 1. In vitro experiments: ① MTT assay and plate cloning assay were used to verify the inhibitory effects of Caerin1.9 and TFMP-Y2 peptides on the proliferation of normal cells and tumor cells in vitro, and to determine the median inhibition concentration (IC50) 50 ) ;② Preparation of peptides by direct labeling with chloramine-T (Ch-T) 131 I-Caerin1.9 and 131I-TFMP-Y2 and the labeling rate, pH value, stability and lipid-water partition coefficient were determined. ③ CCK8 cell proliferation toxicity assay was used to further verify 131 I-Caerin 1.9 and 131 The killing effect of I-TFMP-Y2 on gastric cancer cells in vitro was explored and compared by cell uptake and elution experiments. 131 I-Caerin1.9, 131 I-TFMP-Y2 and control group Na 131 2. In vivo experiments: A subcutaneous gastric cancer tumor model was established in nude mice, and Carein1.9, TFMP-Y2 and 131 I-labeled product: 131 I-Caerin1.9 peptide and 131 I-TFMP-Y2 peptide was injected into the tumor to treat the tumor and the therapeutic effects were compared.

[0012] The results showed that: 1. In vitro experiments: ① Caerin1.9 peptide had a significant inhibitory effect on the proliferation of HGC-27 cells and normal cells, and was concentration-dependent, but TFMP-Y2 peptide had no significant inhibitory effect on gastric cancer HGC-27 cells and human normal gastric mucosal epithelial cells GES-1, and there was a significant statistical difference between the two peptides (P<0.05); the results of the plate cloning experiment showed that the inhibitory effect of Caerin1.9 on HGC-27 cells was positively correlated with its concentration (compared with the control group, P<0.05), and TFMP-Y2 had no significant inhibitory effect on the proliferation of HGC-27 cells. ② Chloramine-T method directly prepared 131 The I-Caerin1.9 labeling rate can reach more than 95% and can remain stable under different conditions. 131 The labeling rate of I-TFMP-Y2 can reach up to 85% and can remain stable. ③ The results of CCK-8 proliferation toxicity experiment are: compared with the simple peptide, the radionuclide labeled peptide product 131 I-Caerin1.9, 131 I-TFMP-Y2 could more effectively inhibit the proliferation of HGC-27, but there was a statistical difference in the inhibitory effects between the two (P<0.05); cell uptake and elution experiments showed that 131 I-Caerin1.9 and 131 Both I-TFMP-Y2 peptides were able to be retained in HGC-27 cells, with 24-hour intracellular binding rates of (17.18±1.13)% and (8.34±1.40)%, respectively. In the in vivo tumor treatment experiment, compared with the control group (PBS group), the subcutaneous tumor volume was significantly reduced after treatment with radionuclide-labeled small molecule peptides, indicating a more significant therapeutic effect, with statistically significant differences (P<0.05).

[0013] The present study shows that the small molecule peptide TFMP-Y2 obtained by Caerin1.9 modification is water-soluble and has no inhibitory effect on normal cell proliferation. Although the simple TFMP-Y2 peptide has no significant inhibitory effect on normal cell proliferation and HGC-27, 131 I-TFMP-Y2 can be stably retained in gastric cancer cells and has significant anti-tumor effects. 131 I-TFMP-Y2 is mainly excreted through the kidney and urinary system, thereby reducing 131 The risk of I-Caerin1.9 radiation-induced liver injury is reduced, thereby overcoming the problems with Caerin1.9 safety and metabolism.

[0014] Specifically, the amino acid sequence of the small molecule polypeptide TFMP-Y2 is YGLHRVLGSAKHAEKL-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 through conventional polypeptide synthesis techniques in the art.

[0015] Furthermore, the iodine-131 labeled small molecule polypeptide TFMP-Y2 is a small molecule polypeptide TFMP-Y2 shown in SEQ ID No. 1, wherein radioactive labeled iodine-131 is bound to the amino acid group.

[0016] The present invention also provides a method for preparing the iodine-131 labeled small molecule polypeptide TFMP-Y2, which comprises using a chloramine-T method to prepare a small molecule polypeptide TFMP-Y2 solution, Na- 131 I solution and chloramine-T solution were shaken and mixed at room temperature, and then separated by chromatography. 131 I-labeled small molecule polypeptide TFMP-Y2 is obtained.

[0017] Furthermore, the TFMP-Y2 and Na- 131 The ratio of I is 40μg:1mCi. 131 I has the highest labeling rate.

[0018] Furthermore, the mass concentration of the TFMP-Y2 solution is 1 mg / mL, and the Na- 131 The ionizing radiation of solution I is 1 mCi (3.7×10 7 The chloramine-T solution must be prepared and used immediately, with a mass concentration of 1 mg / mL.

[0019] The present invention also provides the use of the iodine-131 labeled small molecule polypeptide TFMP-Y2 in the preparation of a drug for treating tumors, specifically the use of the iodine-131 labeled small molecule polypeptide TFMP-Y2 in the preparation of a drug for intratumoral irradiation therapy.

[0020] Preferably, the tumor includes but is not limited to gastric cancer.

[0021] The present invention also provides a tumor treatment drug, which contains the small molecule polypeptide TFMP-Y2 labeled with iodine-131.

[0022] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0023] The dosage of the drugs of the present invention depends on many factors, such as the nature and severity of the condition being treated, the sex, age, weight, and individual response of the patient or animal, the route of administration, and the number of doses. The dose may be administered as a single dose or divided into several doses, such as two, three, or four. The dosage level should be selected based on the specific route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it should be understood that the total daily dosage of the drugs of the present invention should be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level should be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the patient's age, weight, general health, sex, and diet; the time, route, and excretion rate of administration; the duration of treatment; any combination or concurrent medications; and similar factors known in the medical field. For example, it is common practice in the art to start the administration of a dose below that required for the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0024] The present invention also provides the use of iodine-131 labeled caerin1.9 in preparing a drug for treating gastric cancer, specifically the use of iodine-131 labeled caerin1.9 in preparing a drug for treating gastric cancer by internal irradiation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention is based on Caerin1.9 polypeptide, which is modified to enhance its water solubility and reduce its inhibitory effect on normal cells, to obtain a new small molecule polypeptide TFMP-Y2. The simple TFMP-Y2 polypeptide has no significant inhibitory effect on the proliferation of normal cells and HGC-27, but 131 I-TFMP-Y2 can be stably retained in gastric cancer cells and has significant anti-tumor effects. 131 I-TFMP-Y2 is mainly excreted through the kidney and urinary system, thereby reducing 131Therefore, TFMP-Y2 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the survival rates of HGC-27 cells and GES-1 cells under the action of different concentrations of Caerin1.9, P3 peptide, and TFMP-Y2 peptide. A shows the comparison of the survival rates of HGC-27 cells under the action of different concentrations of Caerin1.9 and TFMP-Y2; B shows the comparison of the survival rates of HGC-27 cells under the action of different concentrations of Caerin1.9 and P3 peptide; C shows the comparison of the survival rates of HGC-27 cells under the action of different concentrations of TFMP-Y2 peptide and P3 peptide; D shows the IC values ​​of HGC-27 cells under the action of Caerin1.9, P3 peptide, and TFMP-Y2 peptide. 50 E is the comparison of the survival rates of GES-1 cells under the action of different concentrations of Caerin1.9 and TFMP-Y2; F is the comparison of the survival rates of GES-1 cells under the action of different concentrations of Caerin1.9 and P3 peptide; G is the comparison of the survival rates of GES-1 cells under the action of different concentrations of TFMP-Y2 peptide and P3 peptide; H is the IC values ​​of Caerin1.9, P3 peptide and TFMP-Y2 peptide on GES-1 cells 50 (ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0028] FIG2 is a plate cloning experiment detecting the number of HGC-27 cell colonies under treatment with different concentrations (0, 2, 4, 6, 10, 15 μg / mL) of Caerin1.9 and TFMP-Y2 polypeptides.

[0029] Figure 3 shows the number of HGC-27 cell clones in response to different concentrations of Caerin 1.9 and TFMP-Y2, compared to the control group (0 mg / mL). A shows the results for Caerin 1.9; B shows the results for TFMP-Y2. (ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0030] Figure 4 shows 131 I-Caerin1.9 and 131 The curve of gamma count value measured by I-TFMP-Y2 paper chromatography is plotted. 131 γ counting curve of I-Caerin1.9; B is 131γ counting curve of I-TFMP-Y2; C is 31 I-Caerin1.9, 131 The results of the difference analysis between the I-TFMP-Y2 labeling rates were shown in Table 1 (P < 0.05).

[0031] Figure 5 shows the temperature at 25℃ and 37℃. 131 I-Caerin1.9 and 131 I-TFMP-Y2 sample, 131 I-Caerin1.9 and 131 The radiochemical purity (RCP) of I-TFMP-Y2 mixed with fetal bovine serum (FBS) or normal saline (NS) was stored at room temperature (25°C) and 37°C for different time periods (0, 24, 72h). 131 I-Caerin1.9, 131 The radiochemical purity of I-TFMP-Y2 samples at 25℃ and 37℃ for 0h, 24h and 72h; C~D are 131 I-Caerin1.9, 131 I-TFMP-Y2 was radiochemically pure after mixing with NS at 25℃ and 37℃ for 0h, 24h and 72h; E~F were 131 I-Caerin1.9, 131 I-TFMP-Y2 was radiochemically pure after mixing with FBS at 25°C and 37°C for 0h, 24h, and 72h.

[0032] Figure 6 131 I-Caerin1.9, 131 The results of the cellular uptake and wash-off experiments of I-TFMP-Y2. Among them, A is the HGC-27 cell uptake of Na at different time points (2h, 4h, 6h, 24h). 131 I. 131 I-Caerin1.9 and 131 I-TFMP-Y2 uptake rate; B is elution after 24h incubation, and Na 131 I. 131 I-Caerin1.9 and 131 The binding rate of I-TFMP-Y2 to HGC-27 cells.

[0033] Figure 7 131 I-Caerin1.9, 131 The results of the cell proliferation toxicity test of I-TFMP-Y2. Among them, A, B, and D are the results of the cell proliferation toxicity test of I-TFMP-Y2 with different radioactive concentrations (2000, 5000, 10000, and 20000 KBq / mL). 131I-Caerin1.9, 131 I-TFMP-Y2 and Na 131 Comparison of the survival rate of HGC-27 cells under the action of I; C, E, and F are the survival rates of HGC-27 cells under different drug concentrations (0, 3.3, 7.0, 14.3, and 30.7 μg / mL). 131 I-TFMP-Y2 and TFMP-Y2, 131 Comparison of the survival rates of HGC-27 cells under the action of I-Caerin1.9, Caerin1.9, and Caerin1.9 and TFMP-Y2.

[0034] Figure 8 shows changes in tumor volume, tumor weight, and nude mouse body weight in different treatment groups. A shows changes in tumor volume at different time points after tumor implantation in different treatment groups; B shows weights of isolated tumors in different treatment groups; and C shows changes in nude mouse body weight at different time points after tumor implantation in different treatment groups. (ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0035] FIG9 shows images of tumors isolated from different treatment groups. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0038] (1) Cell lines and cell culture

[0039] Gastric cancer cells (HGC-27) were donated by Lin Shaoqiang's group at the First Affiliated Hospital of Guangdong Pharmaceutical University. Human gastric epithelial cells (GES-1) were purchased from Beina Chuanglian Biotechnology Co., Ltd. HGC-27 cells were cultured in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific, USA) containing 89% RPMI medium 1640 (GIBCO, USA), 10% heat-inactivated fetal bovine serum (FBS, Corning, USA), and 1% penicillin-streptomycin solution (GIBCO, USA).

[0040] (2) Amino acid sequence composition of Caerin1.9 polypeptide, TFMP-Y2 polypeptide, and P3 polypeptide

[0041] Caerin1.9 peptide: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2;

[0042] TFMP-Y2 peptide: YGLHRVLGSAKHAEKL-NH 2；

[0043] P3 peptide: GTELPSPPSVWFEAEF-OH;

[0044] P3 polypeptide, Caerin 1.9, and TFMP-Y2 polypeptide were all synthesized by China Polypeptide Co., Ltd. (Shanghai, China). Their purities were determined to be >95% by reverse-phase HPLC. Caerin 1.9 polypeptide, TFMP-Y2, and P3 polypeptide were dissolved in phosphate buffered saline (PBS, GIBCO, USA) to different concentrations (10 mg / mL, 1 mg / mL, and 0.1 mg / mL) and stored in a −20°C refrigerator.

[0045] (3) Nude mice

[0046] Thirty adult female BALB / C nude mice, 4–6 weeks old and weighing 15–18 g, were purchased from the Guangdong Medical Laboratory Animal Center and maintained at the Animal Resource Center (The First Affiliated Hospital of Guangdong Pharmaceutical University) according to SPF standards. All feed, water, and bedding used were sterile. No animals became ill or died before the end of the experiment. Nude mice were euthanized by cervical dislocation in accordance with the "Regulations on Animal Management" (Ministry of Health of the People's Republic of China). All experiments were conducted in accordance with the guidelines of the Animal Experimentation Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University (Ethics Approval No. FAHGPM20160316).

[0047] The present invention uses the Caerin 1.9 polypeptide as a basis and modifies it to enhance its water solubility and reduce its inhibitory effect on normal cells, resulting in a new small molecule polypeptide, TFMP-Y2 (SEQ ID No. 1). In vitro and in vivo experiments were conducted to investigate the polarity of Caerin 1.9 and TFMP-Y2, and to compare Caerin 1.9 with the modified polypeptide TFMP-Y2 and its iodine-131 labeled product: 131 I-Caerin1.9 peptide and 131 The therapeutic effect of I-TFMP-Y2 polypeptide on gastric cancer is shown in the following examples.

[0048] Data Analysis: All experiments in the following examples were repeated at least three times. All experimental data were analyzed by analysis of variance using GraphPad Prism 9.0.0 software (San Diego, CA, USA), except for the comparison of labeling yields between the two labeled products, which was performed using a t-test. Differences were considered significant when P < 0.05.

[0049] Example 1 MTT assay to investigate the inhibitory effects of Caerin1.9, P3, and TFMP-Y2 peptides on HGC-27 and GES-1 cell proliferation

[0050] 500 mg of MTT powder (Sigma-Aldrich, USA) was dissolved in 100 mL of PBS to a concentration of 5 mg / mL (i.e., 0.5% MTT) and stored in a -20°C refrigerator in the dark. HGC-27 or GES-1 cells in the logarithmic growth phase were obtained and digested with 0.25% trypsin (GIBCO, USA) to prepare a single-cell suspension. The cell count was diluted to 5 × 10 5 Cells were seeded with 100 μL of MTT solution per well in four 96-well plates (Corning, USA) at 37°C in a 5% CO2 incubator for 24 hours. Cells were divided into blank control, Caerin1.9 group, P3 group, and TFMP-Y2 group (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). After cell attachment, the drug was added at the above concentrations. The treated 96-well plates were again incubated in the incubator for 24 hours. Cells were observed under a microscope, and 10 μL of MTT solution was added to each well. The culture was continued for another 4 hours. After terminating the culture, the supernatant was carefully aspirated, and 150 μL of DMSO solution (Sigma-Aldrich, USA) was added to each well. The 96-well plates were shaken at low speed for 10 minutes. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (Thermo Scientific, USA). GraphPad Prism 9.0.0 software was used to calculate the peptide half-maximal inhibitory concentration (IC50) and survival percentage (S%) of each drug.

[0051] The MTT assay results are shown in Figure 1 , and the survival percentage (S%) results are shown in Figures 1A and 1E . Caerin 1.9 peptide had no significant inhibitory effect on HGC-27 cells at concentrations below 5 μg / mL. Its ability to inhibit HGC-27 cell proliferation increased with increasing Caerin 1.9 peptide concentrations. When the Caerin 1.9 peptide concentration exceeded 15 μg / mL, the cell survival rate significantly decreased. At a Caerin 1.9 peptide concentration of 40 μg / mL, the survival rate was (6.35±0.74)%. Caerin 1.9 peptide inhibited GES-1 cell proliferation at concentrations above 10 μg / mL. At a Caerin 1.9 peptide concentration of 40 μg / mL, the survival rate was only (13.80±0.56)%. At high concentrations, the inhibitory and killing effects of both peptides on HGC-27 and GES-1 cells were statistically significant (P<0.05). As shown in Figures 1C and 1G, at a higher concentration (40 μg / mL) of TFMP-Y2 peptide, HGC-27 and GES-1 cells still maintained relatively high survival rates of (93.44±2.53)% and (98.10±2.61)%, respectively. As shown in Figures 1D and 1H, the IC values ​​of Caerin1.9 peptide on HGC-27 cells were significantly higher than those on GES-1 cells. 50 The IC value of Caerin1.9 peptide on GES-1 cells was 16.06 μg / mL. 50 It is 11.48μg / mL.

[0052] Example 2 Plate cloning experiment

[0053] HGC-27 cells in the logarithmic growth phase were digested with 0.25% trypsin (GIBCO, USA) to prepare a single-cell suspension, and the cell count was diluted to 1×10 3 Cells were seeded in 6-well plates (Corning, USA) at 1 mL / well and cultured in a 37°C, 5% CO2 incubator for 24 h. After cell attachment, Caerin 1.9 and TFMP-Y2 peptides were added at different concentrations, resulting in final concentrations of 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 10 μg / mL, and 15 μg / mL. After 24 h, the 6-well plates were exchanged with the medium. Thereafter, the medium was changed every two days, and the cell status and colony number were observed. When the cells in the 0 μg / mL culture wells proliferated to approximately 50, the culture was terminated for 8 days. After terminating the culture, cells were fixed with 4% paraformaldehyde (Sigma, USA) for 20 min, stained with crystal violet solution (Beyotime, China) for 15 min, rinsed three times with PBS, and air-dried. Cell colonies were counted using ImageJ analysis software and analyzed using GraphPad Prism 9.0.0 software.

[0054] The results of the plate cloning assay are shown in Figures 2-3. The number of cell colonies observed under a microscope is shown in Figure 2, demonstrating that the inhibitory effect of Caerin1.9 peptide on HGC-27 cell proliferation is concentration-dependent. Higher Caerin1.9 peptide concentrations increase the inhibitory effect. However, TFMP-Y2 peptide is not concentration-dependent, maintaining a high survival rate in the HGC-27 cell line at higher concentrations. The results of cell colony counting are shown in Figure 3, demonstrating that the number of cell colonies in the plate cloning assay, when treated with Caerin1.9 peptide compared to the control group (0 μg / mL), was statistically significant (P < 0.05) at concentrations of 2 μg / mL, 4 μg / mL, 6 μg / mL, 10 μg / mL, and 15 μg / mL. However, TFMP-Y2 peptide treatment showed no significant inhibitory effect on HGC-27 cells with increasing peptide concentrations, consistent with the results of the MTT assay.

[0055] Example 3 Preparation 131 I-Caerin1.9 peptide, 131 I-TFMP-Y2 polypeptide

[0056] (1) Accurately weigh 10 mg of chloramine-T trihydrate powder and place it in a 15 mL centrifuge tube. Add 10 mL of PBS and mix well to dissolve it. Prepare a 1 mg / mL chloramine-T solution. Store at room temperature in the dark and use it immediately.

[0057] (2) 40 μL of Caerin1.9 peptide (1 mg / mL) was added to a 1.5 mL sterile EP tube (Shanghai Hongsheng, China), followed by the addition of 100 μL of 1 mCi (3.7 × 10 7 Bq)Na 131 I solution (Guangdong Junqi Pharmaceutical, China) and 100 μL of freshly prepared 1 mg / mL chloramine-T solution were added to a total volume of 240 μL, and the mixture was shaken at room temperature for 10 min using a vortex mixer (Thermo Scientific, USA).

[0058] (3) 40 μL of TFMP-Y2 peptide (1 mg / mL) was added to a 1.5 mL sterile EP tube (Shanghai Hongsheng, China), followed by the addition of 100 μL of 1 mCi (3.7 × 10 7 Bq)Na 131 I solution (Guangdong Junqi Pharmaceutical, China) and 100 μL of freshly prepared 1 mg / mL chloramine-T solution were added to a total volume of 240 μL, and the mixture was shaken at room temperature for 10 min using a vortex mixer (Thermo Scientific, USA).

[0059] (4) The radioactivity of the samples was evaluated by thin layer 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-Caerin1.9 and 131 The labeling rate of I-TFMP-Y2 polypeptide was plotted using GraphPad Prism 9.0.0 software, and the labeling rate was obtained by calculating the area under the curve.

[0060] The results are shown in Figure 4. 131 The radiolabeling rate of I-Caerin1.9 was (96.99±0.61)%. 131 The radioactive labeling rate of I-TFMP-Y2 was (86.86±1.72)%, and there was a statistical difference between the two labeling rates (P<0.05).

[0061] Example 4 131 I-Caerin1.9, 131 Stability assay of I-TFMP-Y2

[0062] Will be simple 131 I-Caerin1.9, 131 I-TFMP-Y2 samples and their mixtures with fetal bovine serum and saline were stored at different temperatures (25°C and 37°C) and at different time points (0 h, 24 h, and 72 h). Their radiochemical purity (RCP) was determined by paper chromatography to assess the stability of the two labeled products in different solutions at different temperatures. GraphPad Prism 9.0.0 software was used to calculate RCP and assess their stability.

[0063] The results are shown in Figure 5. 131 I-Caerin1.9 and 131 I-TFMP-Y2 has a high radiochemical purity (RCP) when placed in fetal bovine serum (FBS) and normal saline (NS) at room temperature (25°C) and 37°C for 72 hours. 131 I-Caerin1.9 sample, 131 I-Caerin1.9 and FBS mixture 131 The RCP of the mixture of I-Caerin1.9 and NS stored at 25°C for 72 hours were 91.06% ± 0.51%, 89.22% ± 1.87%, and 85.31% ± 1.01%, respectively; the RCP of the three samples stored at 37°C for 72 hours were 89.34% ± 1.69%, 89.22% ± 3.39%, and 85.43% ± 1.09%, respectively. 131 I-TFMP-Y2 sample,131 I-TFMP-Y2 and FBS mixture and 131 The RCPs of the mixture of I-TFMP-Y2 and NS stored at 25°C for 72 hours were 89.51% ± 2.70%, 89.41% ± 2.87%, and 83.27% ± 3.46%, respectively; the RCPs of the three samples stored at 37°C for 72 hours were 87.54% ± 3.11%, 87.13% ± 0.90%, and 82.99% ± 2.48%, respectively. These results demonstrate that regardless of 131 I-TFMP-Y2, 131 I-Caerin1.9 alone or in mixture with FBS and NS showed good stability at 25°C or 37°C.

[0064] Example 5 Determination of fat-water partition coefficient

[0065] Take two 1.5mL EP tubes and mix 500μL of n-octanol (McLean, China), 500μL of normal saline and 50μL of 131 I-Caerin1.9 or 131 I-TFMP-Y2 was added to separate EP tubes, sealed and vibrated for 2 minutes, and then centrifuged at 4000 rpm for 5 minutes to achieve equilibrium between n-octanol and saline. A 50 μL sample was taken from each of the upper lipid phase and the lower aqueous phase, and the radioactivity count in each tube was measured. This was repeated six times. The lipid-water partition coefficient (Log P) was calculated.

[0066] LogP is an indicator of the solubility of the labeled product in the organic phase or the aqueous phase. A LogP value greater than 0 indicates that the drug tends to be lipid-soluble, and a higher LogP value indicates a higher solubility of the drug in the organic phase. 131 I-Caerin1.9, 131 The lipid-water partition coefficients of I-TFMP-Y2 are shown in Table 1 and Table 2 respectively. 131 I-Caerin1.9, 131 The LogP values ​​of I-TFMP-Y2 were 0.180±0.081 (n=6) and -0.200±0.079 (n=6), respectively. 131 I-Caerin1.9 is fat-soluble, 131 I-TFMP-Y2 is water-soluble.

[0067] Table 1 131 CPM counts and lipid-water partition coefficient of I-Caerin1.9 in lipid and aqueous phases

[0068] Table 2131 CPM counts and lipid-water partition coefficient of ITFMP-Y2 in lipid and aqueous phases

[0069] Example 6 131 I-Caerin1.9, 131 I-TFMP-Y2 cellular uptake experiment

[0070] Take HGC-27 cells in the logarithmic growth phase and dilute the cell count to 1×10 5 Each 24-well plate was divided into three groups: 131 I-caerin1.9 group, 131 I-TFMP-Y2 group and simple 131 Group I, each containing one positive control well and three experimental wells. Cell attachment was observed under a microscope, and the supernatant was aspirated. 500 μL of serum-free medium was added to each well. 2 μL (8 μCi) of the corresponding solution was added to each of the three wells. The 24-well plates were then placed in a cell culture incubator and incubated for 2, 4, 6, and 24 hours before treatment. The supernatant from the experimental wells was discarded, and each well was rinsed twice with PBS. 200 μL of trypsin (GIBCO, USA) was added for digestion, followed by three rinses with PBS. The digestion solution from the experimental wells was collected in the corresponding tube, while all the solution from the positive control wells was collected in the corresponding tube. The radioactivity count in each glass tube was measured using a gamma counter. Drug binding efficiency was calculated using GraphPad Prism.

[0071] The results of the cell uptake experiment are shown in Figure 6A. As time goes by, HGC-27 cells 131 I-Caerin1.9, 131 The uptake of I-TFMP-Y2 showed an increasing trend. At the end of 24 hours, HGC-27 131 I-Caerin1.9, 131 The uptake rates of I-TFMP-Y2 were (17.18±0.87)% and (8.34%±1.40)%, respectively. However, the uptake rates of Na 131 There was almost no uptake of I, and the binding rate at the end of 24 h was only (0.76±0.15)%.

[0072] Example 7 131 I-Caerin1.9, 131 Cell elution experiment of I-TFMP-Y2

[0073] Take HGC-27 cells in the logarithmic growth phase and dilute the cell count to 1×105 Each 24-well plate was divided into three groups: 131 I-caerin1.9 group, 131 I-TFMP-Y2 group and simple 131 Group I, each containing one positive control well and three experimental wells. Cell attachment was observed under a microscope, the supernatant was aspirated, and 500 μL of serum-free medium was added to each well. 2 μL (8 μCi) of the corresponding solution was added to each of the three wells. The 24-well plate was then placed in a cell culture incubator for 24 h. The supernatant was discarded, and each well was rinsed twice with PBS, followed by the addition of 500 μL of serum-free medium. The plates were then placed in an incubator and incubated for 2, 4, 6, and 24 h before treatment. The supernatant from the experimental wells was discarded, and each well was rinsed twice with PBS. 200 μL of trypsin (GIBCO, USA) was added for digestion, followed by three rinses with PBS. The digestion solutions from the experimental wells were collected in corresponding tubes, while all solutions from the positive control wells were collected in corresponding tubes. The radioactivity counts in each tube were measured using a γ counter, and the drug retention rate was calculated using GraphPad Prism 9.0.0 software.

[0074] The results of the cell elution experiment are shown in Figure 6B. 131 I-Caerin1.9 and 131 I-TFMP-Y2 was incubated with HGC-27 cells for 24 hours and then eluted. The binding rate of the drug to HGC-27 cells remained at a high level at each time point (2, 4, 6, and 24 hours) and then slowly decreased. 131 I-Caerin1.9 and 131 The binding rates of I-TFMP-Y2 and HGC-27 cells were (75.84±1.13)% and (89.44±7.08)%, respectively. 131 I had almost no uptake, so the cell binding rate was only (4.98±1.08)% 24 hours after elution.

[0075] The above cell uptake and elution experiments showed that both radiolabeled products could be taken up by HGC-27 cells and were relatively stably retained in the cells.

[0076] Example 8 131 I-Caerin1.9, 131 Toxicity test of I-TFMP-Y2 on HGC-27 cell proliferation

[0077] Take HGC-27 cells in the logarithmic growth phase and dilute the cell count to 5×105 The cells were seeded at 100 μL / well in two 96-well plates (Corning, USA) and cultured for 24 hours (5% CO 2 , 37° C. incubator). 131 I-Caerin1.9, 131 I-TFMP-Y2 and Na 131 The drug concentrations for the Caerin 1.9 peptide and TFMP-Y2 peptide groups were 2000, 5000, 10,000, and 20,000 KBq / mL, respectively. Three replicate wells were set up for each concentration, and three untreated control groups were also set up. Cultures were continued for 24 hours, after which 10 μL of CCK-8 (DOJINDO, Japan) was added to each well and incubated in the dark for 4-6 hours. Cell viability was calculated using enzyme-linked immunosorbent assay (ELISA) at 450 nm using GraphPad Prism 9.0.0 software.

[0078] The experimental results are shown in Figure 7. The CCK-8 experiment shows that 131 I-Caerin1.9 and 131 With the increase of I-TFMP-Y2 radioactive concentration, the cytotoxic effect gradually increased and the cell survival rate gradually decreased. 131 When the radioactive concentrations of I-Caerin1.9 were 2500, 5000, 10000, and 20000 KBq / mL, the survival rates of HGC-27 cells were (79.94±0.88)%, (62.11±1.61)%, (42.02±3.09)%, and (18.87±0.12)%, respectively. At the same drug concentrations (3.3μg / mL, 7.0μg / mL, 14.3μg / mL, and 30.7μg / mL), the survival rates of Caerin1.9 peptide alone were significantly higher than those of Caerin1.9 peptide alone. 131 I-TFMP-Y2 had a weak inhibitory effect on HGC-27 cells, and the differences were statistically significant (P < 0.05). 131 When the radioactive concentrations of I-TFMP-Y2 were 2500, 5000, 10000, and 20000 KBq / mL, the survival rates of HGC-27 cells were (94.26±3.52)%, (77.47±1.57)%, (58.63±2.60)%, and (41.55±2.91)%, respectively. Similarly, at the same drug concentrations, TFMP-Y2 alone had no significant inhibitory effect on HGC-27 cells, and the differences were statistically significant (P<0.05). 131 I did not show an inhibitory effect on the proliferation of HGC-27. At different concentrations, 131I-Caerin1.9 and 131 The cytotoxic effect of I-TFMP-Y2 on HGC-27 cells showed significant statistical differences at high radioactive concentrations (P>0.05).

[0079] Example 9 Establishment of nude mouse subcutaneous tumor model and in vivo treatment

[0080] 100 μL of HGC-27 cells (3 × 10 6 Tumors were inoculated subcutaneously in the axilla of nude mice to establish a subcutaneous tumor model. Tumors were used for experiments when their diameter approached 3–4 mm. Tumor size was monitored every 2 days using a digital caliper (Mitutoyo, Japan, CD-15APX), and tumor volume was calculated according to the following formula: volume = 0.5 × width 2 × length.

[0081] To minimize the thyroid 131 Three days before the first in vivo treatment, all nude mice were fed with 0.1% potassium iodide (McLean, China) to block the thyroid gland. Tumor-bearing nude mice were randomly divided into six groups (n = 4 per group): PBS group, Caerin1.9 group, TFMP-Y2 group, 131 Group I, 131 I-Caerin1.9 group, 131 The nude mice were treated with the following methods: the volume of drug injected into the tumor was 100 μL each time; the PBS group (control group) was injected with pure PBS; the Caerin1.9 group was injected with 10 μg of Caerin1.9 peptide; and the TFMP-Y2 group was injected with 10 μg of TFMP-Y2 peptide. 131 Group I was injected with 200 μCi of Na 131 I solution, 131 The I-Caerin1.9 group was injected with 10 μg Caerin1.9 peptide and 200 μCi Na- 131 I mixed solution, 131 The I-TFMP-Y2 group was injected with 10 μg TFMP-Y2 peptide and 200 μCi Na- 131 The mixture of 1 and 2 was injected once every 2 days for a total of 4 times. On the 5th day after the last treatment, the nude mice were euthanized, and the tumors were isolated and weighed. Statistical analysis of nude mouse body weight, tumor volume, and tumor weight was performed using GraphPad Prism 9.0.0 software.

[0082] The experimental results are shown in Figures 8-9. There was no statistically significant difference in the tumor volume of the nude mice in each group at the beginning of treatment (P>0.05). The weight of the nude mice in each group showed no significant decrease compared with that before treatment. Before euthanasia, the weight of the nude mice in each group was as follows: PBS group 17.73±0.76g, Caerin1.9 group 17.20±0.31g, TFMP-Y2 group 15.91±1.06g, 131 Group I: 14.70±0.48g, 131 I-Caerin1.9 group 14.43±0.69g, and 131 The I-TFMP-Y2 group was 15.86±0.58g. As shown in Figure 8A and B, the PBS group 131 Group I, 131 The tumor volumes of the I-TFMP-Y2 group, Caerin1.9 group, and TFMP-Y2 group increased compared with those before the first treatment. 131 The tumor volume of the I-Caerin1.9 group was significantly smaller than that before treatment. As shown in Figure 8C, before treatment, the tumor volume of the PBS group was 16.08±0.72mm 3 , Caerin1.9 group 15.10±0.80mm 3 TFMP-Y2 group: 15.61±0.48mm 3 、 131 Group I: 15.50±0.31mm 3 、 131 I-Caerin1.9 group: 16.77±0.64mm 3 and 131 I-TFMP-Y2 group: 16.47±0.35mm 3 As shown in Figure 8B , after treatment and before euthanasia, the tumor volumes of nude mice in each group were as follows: PBS group: 40.066±2.79mm 3 , Caerin1.9 group 31.849±3.34mm 3 TFMP-Y2 group: 40.93±1.08mm 3 、 131 Group I: 39.74±1.84mm 3 、 131 I-Caerin1.9 group: 13.75±1.08mm 3 and 131 I-TFMP-Y2 group: 19.17±1.51mm 3 As shown in Figure 8D and Figure 9, after the nude mice were euthanized, the tumors were isolated, weighed, and photographed. The tumor weights of each group were as follows: PBS group 16.55±1.90 mg, Caerin1.9 group 11.03±0.82 mg, TFMP-Y2 group 16.50±2.32 mg, 131Group I: 15.95±1.13mg, 131 I-Caerin1.9 group 3.75±0.79mg and 131 I-TFMP-Y2 group 8.90±1.84mg. 131 I-Caerin1.9 can inhibit the growth of gastric cancer in vivo. In contrast, 131 The inhibitory effect of I-TFMP-Y2 was relatively weak. At the end of treatment, the inhibitory effect of I-TFMP-Y2 was relatively weak. 131 I group, Caerin1.9 group, TFMP-Y2 and 131 Compared with the I-TFMP-Y2 group, both tumor volume and tumor weight showed statistically significant differences (P < 0.05); and consistent with the CCK-8 experiment, 131 I-Caerin1.9 group and 131 There were statistically significant differences in tumor volume and tumor weight between the I-TFMP-Y2 group (P < 0.05).

[0083] The present invention demonstrated that Caerin 1.9 exhibited concentration-dependent antitumor activity against both gastric cancer cells and normal gastric mucosal epithelial cells through MTT assays, whereas the modified polypeptide TFMP-Y2 had no significant effect on the growth of these cells. A plate colony formation assay further demonstrated that Caerin 1.9 exhibited a significant inhibitory effect against gastric cancer cells.

[0084] The present invention uses the chloramine-T method to prepare two polypeptides 131 I-labeled product, i.e. 131 I-Caerin1.9 and 131 I-TFMP-Y2, the former has a labeling rate greater than 95%, and the latter has a labeling rate greater than 85%, and it is proven that both have high stability in physiological saline and fetal bovine serum at different temperatures (25°C and 37°C). 131 The labeling rate of I-TFMP-Y2 is lower than before, which may be due to the change of chemical structure. 131 The amount of amino acids bound by I is reduced, but the labeling rate can be increased to more than 90% by physical purification. The results of the lipid-water partitioning experiment show 131 I-Caerin1.9 is lipid soluble, while the modified peptide 131 I-TFMP-Y2 is water-soluble, indicating 131 I-Caerin1.9 may be mainly excreted through the liver. 131 I-TFMP-Y2 may be mainly excreted through the kidneys, thereby reducing the risk of radiation-induced liver injury. 131 I is 131I-Caerin1.9 and 131 The results of the control group of I-TFMP-Y2 showed that as the incubation time increased, 131 I-Caerin1.9, 131 The binding ability of I-TFMP-Y2 in HGC-27 cells gradually increased, and the binding rate was significantly higher than that of Na 131 Group I, confirmed 131 I-Caerin1.9, 131 I-TFMP-Y2 can be taken up by HGC-27 cells and stably retained in HGC-27 cells, but 131 The binding ability of I-TFMP-Y2 in gastric cancer cells is 131 I-Caerin 1.9 is weak. HGC-27 cells hardly take up Na 131 I, mainly because HGC-27 cells do not express sodium iodide symporter (NIS). In the cell elution experiment, 131 After elution for 2h, 4h, 6h and 24h, I-Caerin1.9 131 I-Caerin1.9, 131 The binding rate of I-TFMP-Y2 in HGC-27 cells gradually decreased, and the binding rates at 24 h were (51.55±0.84)% and (62.30±0.69)%, respectively. 131 The 24h binding rate of group I was (4.78±1.08)%, indicating 131 I-Caerin1.9 and 131 The I-TFMP-Y2 polypeptide has good binding ability and long retention time in cells. 131 I-Caerin1.9 can be taken up by lung cancer (A549 cells) and is mainly enriched in the cytoplasm. It can be preliminarily inferred that 131 I can also enter gastric cancer cells after binding to Caerin1.9 and TFMP-Y2 peptides. 131 To investigate whether Caerin 1.9 and TFMP-Y2 peptides can jointly exert an inhibitory effect in HGC-27 cells, a cell proliferation toxicity test (CCK8) was performed. The results of the cell proliferation toxicity test showed that the two labeled products had a toxic killing effect on HGC-27 cells and were more effective than the simple Caerin 1.9, TFMP-Y2 peptides and Na 131 I has a stronger anti-tumor effect. It is speculated that Caerin1.9 and TFMP-Y2 peptides can carry 131 I enters HGC-27 and stably binds to the cells, and enhances 131 The radiation effect of I or 131I combined with the peptide to produce an inhibitory effect on HGC-27 cells; 131 I could not enter HGC-27 cells, which was consistent with the results of the previous uptake and elution experiments.

[0085] In the in vivo experiment, the present invention established a nude mouse subcutaneous transplantation tumor model of HGC-27 cells. With the increase of the number of treatments, the tumor volume gradually decreased. The results showed that at the end of treatment, the tumor volume was significantly reduced compared with the PBS group or Na 131 Compared with group I, Caerin1.9, 131 I-caerin1.9, 131 The tumor volumes and weights of the three nude mice groups were significantly different (P<0.05), which was consistent with the results of MTT experiment, cloning experiment and cell proliferation toxicity experiment. 131 I-Caerin1.9, 131 There were also differences in the therapeutic effects of the two I-TFMP-Y2 groups, which was consistent with the results of the cell proliferation toxicity experiment. 131 I works together with polypeptides, which is simpler 131 I has a better radiation effect. During the experiment, the weight of nude mice in each group remained stable, and no illness or death occurred, indicating that both polypeptides have good biosafety.

[0086] In summary, the experimental results of the present invention show that 131 I-Caerin1.9 and modified peptides 131 I-TFMP-Y2 has tumor targeting properties and can bind to gastric cancer (HGC-27) cells and stably retain in tumor cells, and has a significant tumor cell cytotoxicity and killing effect. In addition, the simple TFMP-Y2 peptide has no significant killing effect on normal cells and tumors, thereby avoiding the inhibitory effect of high concentrations of Caerin1.9 on normal cell proliferation. 131 I-TFMP-Y2 peptide is water-soluble and is mainly excreted through the kidneys, thereby reducing 131 The risk of I-Caerin1.9 radiation-induced liver injury further confirms that TFMP-Y2 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. A small molecule polypeptide TFMP-Y2, characterized in that: Its amino acid sequence is YGLHRVLGSAKHAEKL-NH2.

2. An iodine-131 labeled small molecule polypeptide TFMP-Y2, characterized in that: The amino acid sequence of the small molecule polypeptide TFMP-Y2 is YGLHRVLGSAKHAEKL-NH2, and radioactive labeled iodine-131 is bound to its amino acid group.

3. The method for preparing the iodine-131 labeled small molecule polypeptide TFMP-Y2 according to claim 2, characterized in that: In order to use the chloramine-T method, the small molecule peptide TFMP-Y2 solution, Na- 131 I solution and chloramine-T solution were shaken and mixed at room temperature, and then separated by chromatography. 131 I-labeled small molecule polypeptide TFMP-Y2 is obtained.

4. The preparation method according to claim 3, characterized in that The TFMP-Y2 and Na- 131 The ratio of I is 40μg:1mCi.

5. Use of the iodine-131 labeled small molecule polypeptide TFMP-Y2 according to claim 2 in the preparation of a drug for treating tumors.

6. The application according to claim 5, characterized in that The tumor is gastric cancer.

7. A tumor treatment drug, characterized in that: Contains the iodine-131 labeled small molecule polypeptide TFMP-Y2 according to claim 2.

8. The medicine according to claim 7, characterized in that Pharmaceutically acceptable excipients are also included.

9. Use of iodine-131 labeled caerin 1.9 in the preparation of a drug for treating gastric cancer, characterized in that: The amino acid sequence of the iodine-131 labeled caerin1.9 is GLFGVLGSIAKHVLPHVVPVIAEKL-NH2, and radioactive labeled iodine-131 is bound to the amino acid group.

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