Iodine-131-labeled small molecule polypeptide TCMP-y1, preparation method therefor and use thereof

Abstract

Provided are an iodine-131-labeled small molecule polypeptide TCMP-Y1, a preparation method therefor, and a use thereof. TCMP-Y1 is a small-molecule polypeptide isolated and identified from toad skin secretions in the early stage and has an amino acid sequence of YFKAWGWPIDDATTEHL-NH2. The polypeptide TCMP-Y1 has no obvious proliferation inhibitory effect on prostate cancer cells. However, iodine-131-labeled 131I-TCMP-Y1 can bind to prostate cancer cells and is stably retained within the prostate cancer cells, and has the inhibitory effects on tumor cell proliferation and tumor growth of the prostate cancer cells both in vivo and in vitro. In addition, the water-soluble 131I-TCMP-Y1is mainly excreted by means of the renal-urinary system, thereby reducing the risk of radiation-induced liver injury. The TCMP-Y1 is a small-molecule polypeptide having excellent nuclide-carrying capability, improves drug safety and tumor targeting, and can be used as a potential drug for internal irradiation therapy for tumors.

Description

An iodine-131 labeled small molecule polypeptide TCMP-Y1, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an iodine-131 labeled small molecule polypeptide TCMP-Y1, its preparation method, and its application. Background Technology

[0002] Prostate cancer poses a serious threat to men's health, ranking second in incidence and fifth in mortality worldwide. The primary treatment for early-stage or localized prostate cancer is radical surgery and androgen deprivation therapy (ADT), which often achieves a cure. However, as prostate cancer progresses, almost all patients who were sensitive to androgen deprivation therapy in the early stages develop hormone resistance, i.e., castration-resistant prostate cancer (CRPC). Related studies indicate that due to the insidious onset of prostate cancer, the lack of specific early symptoms, and the current lack of effective screening programs for prostate cancer in my country, most patients only seek medical attention when they experience discomfort, or are diagnosed at an advanced stage, missing the optimal window for surgical treatment. Therefore, there is an urgent clinical need to develop new treatment methods for prostate cancer.

[0003] Radiation therapy is an indispensable part of prostate cancer treatment, and most prostate cancer patients require it, including external beam radiation therapy (EMB) and internal radiation therapy (IRT). IRT utilizes the property of radioactive isotopes or their labeled compounds to target and accumulate in diseased tissue. The radioactive isotope is introduced into the body, and the alpha and beta rays emitted by it produce sufficient ionizing radiation biological effects in the lesion, thereby inhibiting or destroying the diseased tissue. This leads to metabolic disorders, loss of reproductive capacity, cellular senescence, and even apoptosis of the diseased cells. The alpha and beta rays emitted by radioactive isotopes have high energy and short range; the key to this treatment is to maximize tumor damage while protecting healthy tissue, thus achieving a high therapeutic effect.

[0004] Nuclear medicine has evolved to the point where many mature radiopharmaceuticals have been developed for treatment, including... 131 I - Treatment of thyroid cancer 89 Sr treatment for bone metastases and relief of bone pain 32 P is used to treat polycythemia vera. 131 I-MIBG treatment for pheochromocytoma211 At-tamoxifen is used to treat breast cancer, among other conditions. In recent years, radiolabeled peptides have become a popular trend due to their advantage of rapid clearance from plasma and accumulation in target tissues, resulting in lower myelotoxicity. Examples include octreotide, an analogue of somatostatin. 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 exert their antibacterial, antiviral, antifungal, and anticancer effects by disrupting the membrane integrity of bacteria, viruses, and fungi or inhibiting certain cellular functions. Notably, among 2981 databases of AMPs, over 1000 use amphibians as hosts, while only 200 use humans. A variety of host defense peptides have been isolated from the skin gland secretions of amphibians, such as the Australian tree frog and toad, most of which possess antitumor and antibacterial activities. The host defense peptides Caerin 1.1 and Caerin 1.9, isolated from the glandular secretions of the Australian tree frog (Litoria), have been shown to possess various biological activities, including antibacterial, antiviral, antifungal, anticancer, and immunomodulatory effects. Previous research by the inventors' research team has demonstrated that, at certain concentrations, Caerin 1.1 and Caerin 1.9 peptides have inhibitory effects on various human tumor cells, including cervical cancer cells, breast cancer cells, and undifferentiated thyroid cancer cells. Regarding Caerin 1.1 peptide, Caerin 1.9 peptide, and their iodine-131 labeling: 131 I-Caerin1.1 and 131 The effects of I-Caerin 1.9 on prostate cancer have not yet been reported. Furthermore, developing and exploring more naturally derived host defense peptides with anti-tumor effects is of great significance. Summary of the Invention

[0006] The purpose of this 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 objective of this invention is to provide an iodine-131 labeled small molecule polypeptide TCMP-Y1.

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

[0009] A 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 a medicament for treating tumors.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] Previous studies have demonstrated that Caerin 1.1 peptide and Caerin 1.9 peptide have inhibitory effects on various tumors, including lung cancer and melanoma, and that iodine-131 labeled... 131 I-Caerin 1.1 peptide, 131 I-Caerin 1.9 peptide exhibits stronger antitumor activity. Caerin 1.1 peptide, Caerin 1.9 peptide, and their iodine-131-labeled derivatives are also mentioned. 131 I-Caerin1.1 and 131 The effects of I-Caerin 1.9 on prostate cancer have not yet been reported. TCMP-Y1 polypeptide is a small molecule polypeptide isolated and identified from toad skin secretions, and like Caerin 1.1 and Caerin 1.9, it is a polypeptide derived from amphibians. This invention, through research and comparison... 131 I-Caerin 1.1 131 I-Caerin1.9 and 131 The therapeutic effect of I-TCMP-Y1 in prostate cancer cell xenografts (DU145) was explored and compared. 131 I-Caerin 1.1 131 I-Caerin1.9 and 131 In vitro inhibitory effect of I-TCMP-Y1 on the proliferation of prostate cancer cells DU145 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 using the MTT assay and the median inhibition concentration (IC50) was determined. 50 The antitumor effects of Caerin1.1 and Caerin1.9 peptides were verified using a plate colony formation assay.

[0013] (2) Prepared using chloramine-T (Ch-T) labeling method 131 I-Caerin 1.1 peptide, 131 I-Caerin 1.9 peptide and 131 The labeling rate, lipid-water partition coefficient, and stability of the I-TCMP-Y1 peptide were determined.

[0014] (3) Through cell uptake and elution experiments, compare whether prostate cancer cells DU145 can take up cells. 131 I-Caerin 1.1 131 I-Caerin1.9 and 131 I-TCMP-Y1 and their intracellular retention were further verified using CCK8 cell proliferation and toxicity assays. 131 I-Caerin 1.1 131 I-Caerin1.9 and 131 Differences in the killing effect of I-TCMP-Y1 on prostate cancer cells DU145 in vitro.

[0015] (4) Establish a nude mouse model of prostate cancer cells (DU145 cell line) and perform... 131 I-Caerin 1.1 131 I-Caerin 1.9 131 Intratumoral injection of I-TCMP-Y1, Caerin1.1 peptide, Caerin1.9 peptide and TCMP-Y1 peptide was used to treat tumors and compare their differences in in vivo antitumor effects.

[0016] The results showed that: (1) Both Caerin 1.1 and Caerin 1.9 peptides significantly inhibited the proliferation of prostate cancer cells, and this effect was enhanced with increasing concentration. The IC50 values ​​of the two peptides were [missing data]. 50 The concentrations were 10.87 μg / mL and 14.50 μg / mL, respectively; while the TCMP-Y1 peptide 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 products was greater than 90%; and 131 I-Caerin 1.1 131 I-Caerin 1.9 is lipid-soluble, while 131 I-TCMP-Y1 is water-soluble, and all three exhibit high stability under different in vitro environments. 131 I-Caerin 1.1 131 I-Caerin 1.9 has higher stability.

[0018] (3) Cell uptake and elution experiments proved 131 I-Caerin 1.1 131 I-Caerin 1.9 131 I-TCMP-Y1 can be taken up by DU145 cells and retained relatively stably within the cells. Compared with simple peptides, under in vivo or in vitro conditions, 131 I-Caerin 1.1131 I-Caerin 1.9 131 I-TCMP-Y1 both have better ability to inhibit tumor cell proliferation and tumor growth.

[0019] The present invention studies 131 I-Caerin 1.1 peptide, 131 I-Caerin 1.9 peptide and 131 I-TCMP-Y1 peptides can bind to and stably retain prostate cancer cells, and simultaneously inhibit tumor cell proliferation and growth in both in vivo and in vitro. Furthermore, they are water-soluble. 131 I-TCMP-Y1 is primarily excreted through the renal urinary system, thereby reducing the risk of radiation-induced liver injury. This invention provides Caerin 1.1 peptide, Caerin 1.9 peptide, and their iodine-131-labeled derivatives. 131 I-Caerin 1.1 peptide, 131 A novel application of I-Caerin1.9 peptide in the treatment of prostate cancer, and also provides a new small molecule peptide 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 using conventional polypeptide synthesis techniques in the field.

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

[0022] This invention also provides a method for preparing the iodine-131 labeled small molecule polypeptide TCMP-Y1, which involves using the chloramine-T method to mix the small molecule polypeptide TCMP-Y1 solution with Na- 131 Solution I and chloramine-T solution were mixed by shaking 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 40 μg: 1 mCi. 131 I had the highest marking rate.

[0024] Furthermore, the TCMP-Y1 solution has a mass concentration of 1 mg / mL, and the Na- 131 The ionizing radiation of solution I is 1 mCi (3.7 × 10⁻⁶).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 application of the iodine-131 labeled small molecule polypeptide TCMP-Y1 in the preparation of drugs for treating tumors, specifically the application of the iodine-131 labeled small molecule polypeptide TCMP-Y1 in the preparation of drugs for intratumoral irradiation therapy.

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

[0027] The present invention also provides a tumor treatment drug containing an iodine-131 labeled small molecule polypeptide TCMP-Y1.

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

[0029] The dosage of the medicament of this 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 frequency of administration. The dosage can be administered in a single dose or in several doses, such as two, three, or four doses. The dosage level must be selected based on the specific route of administration, the severity of the condition being treated, and the patient's condition and medical history. However, it should be understood that the total daily dosage of the medicament of this invention must be determined by the attending physician within the bounds of reliable medical judgment. For any specific patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the disorder being treated and its severity; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the time of administration, route of administration, and excretion rate; the duration of treatment; other medications used in combination or concurrently; and similar factors known in the medical field. For example, it is practiced in the art to start with a dosage below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0030] This invention also provides the use of Caerin1.1 polypeptide or iodine-131-labeled Caerin1.1 polypeptide in the preparation of drugs 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 has radioactively labeled iodine-131 bound to the amino acid groups of the Caerin1.1 polypeptide.

[0031] The present invention also provides the use of Caerin1.9 polypeptide or iodine-131 labeled Caerin1.9 polypeptide in the preparation of drugs 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 has radioactively labeled iodine-131 bound to the amino acid group of the Caerin1.9 polypeptide.

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

[0033] This invention provides a novel small molecule polypeptide, TCMP-Y1, which was isolated and identified from toad skin secretions and, like Caerin 1.1 and Caerin 1.9, is an amphibian-derived polypeptide. This invention demonstrates... 131 I-Caerin 1.1 peptide, 131 I-Caerin 1.9 peptide and 131 I-TCMP-Y1 peptides can bind to and stably retain prostate cancer cells, inhibiting tumor cell proliferation and growth both in vivo and in vitro. They are also water-soluble. 131 I-TCMP-Y1 is primarily excreted through the renal urinary system, thereby reducing the risk of radiation-induced liver injury. This invention provides a novel small molecule polypeptide, TCMP-Y1, with antitumor activity, offering more options for cancer treatment. Simultaneously, this invention expands upon the use of Caerin 1.1 polypeptide, Caerin 1.9 polypeptide, and... 131 I-Caerin 1.1 peptide and 131 Scope of application of I-Caerin1.9 peptide. Attached Figure Description

[0034] Figure 1 shows the effects of different concentrations of Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide on the proliferation of DU145 cells and their IC50 values. Figures 1A, 1B, and 1C: Survival rates of DU145 cells under different concentrations of Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide. Figure 1D: IC50 values ​​of Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide on DU145 cells. 50 Values. (ns P>0.05; **P<0.01; ****P<0.0001).

[0035] Figure 2 shows clones of DU145 cells after treatment with different concentrations of Caerin1.1 and Caerin1.9 peptides.

[0036] Figure 3 shows the inhibition of DU145 cell proliferation by Caerin 1.1 and Caerin 1.9 peptides. The number of cell clones under different drug concentrations was compared with the control group (drug concentration 0 μg / mL). Statistical analysis was performed using Image-J software. (ns P>0.05; *P<0.05; **P<0.01; ****P<0.0001).

[0037] Figure 4 is 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 A graph of the gamma count value measured by paper chromatography using I-TCMP-Y1. Figures 4A, 4B, and 4C respectively show... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The curve plotted by measuring the γ count value using paper chromatography with I-TCMP-Y1 showed that the labeling rate of both was greater than 90%.

[0038] Figure 5 shows the radiochemical purity of the three labeled products under different environments. 131 I-Caerin 1.1 131 I-Caerin 1.9 131 I-TCMP-Y1 131 I-Caerin 1.1 or 131 I-Caerin 1.9 or 131 Radiochemically pure (RCP) of a mixture of I-TCMP-Y1 with fetal bovine serum or physiological saline stored at room temperature (25°C) and 37°C for different times (0h, 24h, 72h).

[0039] Figure 6 shows the results of cell uptake and elution experiments. Figure 6A shows the DU145 cells at different time points (2h, 4h, 6h, 24h) and Na⁻. 131 I, 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The binding rate of I-TCMP-Y1; Figure 6B shows the Na- ionization rate at different time points (2h, 4h, 6h, 24h) after 24h incubation and elution of the drug with DU145 cells. 131 I, 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The binding of I-TCMP-Y1 to DU145 cells.

[0040] Figure 7 shows the results of the cytotoxic proliferation experiment. 7A, 7B, and 7C represent the results of the reaction with Na+, etc. 131 I. Comparison of different radioactive concentrations (2500 KBq / mL, 5000 KBq / mL, 10000 KBq / mL, 20000 KBq / mL) 131 I-Caerin 1.1 131 I-Caerin 1.9 131 Comparison of DU145 cell survival rates under I-TCMP-Y1 treatment; Figures 7D, 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-Caerin 1.1 and Caerin 1.1 131 I-Caerin 1.9 and Caerin 1.9 (7 μg / mL, 14 μg / mL, 28 μg / mL, 56 μg / mL) 131 Comparison of DU145 cell survival rates under I-TCMP-Y1 and TCMP-Y1 treatment; Figures 7G, ​​7H, and 7I represent different radioactive concentrations. 131 I-Caerin1.1 and 131 Between I-Caerin 1.9 131 I-Caerin1.1 and 131 Between I-TCMP-Y1 131 I-Caerin1.9 and 131 Comparison of DU145 cell viability between I-TCMP-Y1; Figures 7J, 7K, and 7L represent comparisons of DU145 cell viability between different drug concentrations of Caerin1.1 and Caerin1.9, Caerin1.1 and TCMP-Y1, and Caerin1.9 and TCMP-Y1, respectively. (ns P>0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0041] Figure 8 shows the changes in tumor volume, tumor weight, and body weight of nude mice in different treatment groups. A represents the tumor volume of DU145-bearing nude mice during treatment (top); B represents the tumor volume of DU145-bearing nude mice during treatment (bottom); C represents the body weight of nude mice during treatment with DU145; and D represents the weight of the isolated tumor during treatment with DU145. (ns P>0.05; **P<0.01; ****P<0.0001).

[0042] Figure 9 shows images of tumors isolated from different treatment groups.

[0043] Figure 10 shows the H&E staining results of tumor sections from different treatment groups and the statistical results of differences in the area of ​​cell degeneration and necrosis. In the figure, A shows the H&E staining of tumor sections from different treatment groups; B shows the statistical results of differences in the area of ​​cell degeneration and necrosis from different treatment groups. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0045] Unless otherwise specified, all 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. They were cultured in a Thermo incubator (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] Caerin 1.1 peptide: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2;

[0050] Caerin 1.9 peptide: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2;

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

[0052] Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide were synthesized and purified by Shanghai Qiangyao Biotechnology Co., Ltd. The purity of all three peptides was >95% as determined by high-performance liquid chromatography (HPLC). The structural formulas of Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide are shown below:

[0053] The Caerin1.1 peptide, Caerin1.9 peptide, and TCMP-Y1 peptide were dissolved in phosphate buffer (PBS, GIBCO, USA) to form a peptide solution of 1 mg / mL and stored at -20°C.

[0054] (3) Nude mice

[0055] BALB / C male nude mice aged 4 - 6 weeks and specific pathogen free (SPF) were purchased from Guangdong Provincial Center for Medical Laboratory Animals (license number: SCYK(Guangdong)2018 - 0002), and were raised under SPF conditions in the SPF experimental animal center of the First Affiliated Hospital of Guangdong Pharmaceutical University (license number: SYXK(Guangdong)2017 - 0124). Sterile feed, water source, and an environment with 12 - hour light + 12 - hour dark alternation were provided. At the end of the experiment, the mice were euthanized by cervical dislocation according to the "Implementing Rules for the Administration of Medical Laboratory Animals" (Order No. 55) of the Ministry of Health of the People's Republic of China.

[0056] In this invention, by researching and comparing 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 I - TCMP - Y1, the inhibitory effect on the proliferation of prostate cancer cell DU145 in vitro and the therapeutic value for prostate cancer in vivo were studied. The specific experiments are as follows in the following examples.

[0057] All data statistical analysis was performed using SPSS 26.0 software, and the data was described by mean ± standard deviation (mean ± SD). A t - test was used between two samples to check whether the data conforms to a normal distribution. ANOVA variance analysis was used for comparisons between three or more groups. GraphPad 9.0 was used to draw and present the experimental result graphs. When P < 0.05, it was considered that there was a statistical difference.

[0058] Example 1 MTT experiment to explore the inhibitory effect of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP - Y1 polypeptide on the proliferation of DU145 cells

[0059] In this example, the survival rate of DU145 cells was detected by the MTT method, and then the toxic proliferation effects of Caerin1.1 polypeptide, Caerin1.9 polypeptide and TCMP - Y1 polypeptide on prostate cancer cell DU145 in vitro were studied. The specific method steps are as follows:

[0060] (1) DU145 cells with good growth status and a cell density of 80% - 90% were selected for the MTT experiment. After disinfecting, rinsing, digesting, counting, and centrifuging the DU145 cells, they were seeded into a 96 - well plate. The cell suspension of the above - mentioned DU145 cells was added to each well except for the outermost ring, and the cell density was 5×10 3100 μL / well; add 100 μL of sterile PBS to the outermost well. After plating, incubate the 96-well plate at 37°C with 5% CO2 for 24 hours; add the drug when the cell density is approximately 80%.

[0061] (2) Melt the pre-prepared 1 mg / mL Caerin 1.1 peptide, Caerin 1.9 peptide and TCMP-Y1 peptide at room temperature for later use;

[0062] (3) A zeroing group (containing only 100 μL PBS); a control group (containing only 100 μL DU145 cell suspension); and three experimental groups (containing 100 μL DU145 cell suspension and the drug): Caerin 1.1 peptide group, Caerin 1.9 peptide group, and TCMP-Y1 peptide group. Eight concentration gradients were set up for each of the three experimental groups: 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. Each group had three replicates, and the experiment was repeated three times.

[0063] (4) After adding the drug according to the above method, place the 96-well plate in an incubator for further culture;

[0064] (5) After 24 hours, add 10 μL of MTT solution to each well, then wrap the 96-well plate with tin foil to protect it from light, place it in an incubator, and continue to incubate.

[0065] (6) After 4 hours, terminate the culture, discard the supernatant, add 150 μL of DMSO to each well, and place on a shaker to shake at low speed for 10 minutes.

[0066] (7) Measure the OD value of each well using an ELISA reader (wavelength 570nm). Calculate the cell viability rate = (average OD value of each experimental group - average OD value of the zeroing group) / (average OD value of the control group - average OD value of the zeroing group) × 100%.

[0067] (8) Using the cell viability of the three drugs obtained in the above MTT experiment, the IC50 of the three peptides was calculated using GraphPad Prism 9.0.0 software. 50 .

[0068] The results of the MTT assay are shown in Figure 1, specifically Figures 1A, 1B, and 1C. When the concentrations of Caerin1.1 and Caerin1.9 peptides were 15 μg / mL, the cell viability was significantly reduced to (35.28±1.36)% and (41.86±0.91)%, respectively. When the concentration reached 20 μg / mL, the cell viability was only (20.32±0.38)% and (33.18±2.66)%, respectively. However, when the concentration of TCMP-Y1 peptide reached 20 μg / mL, the cell viability remained high at (100.74±2.16)%, significantly higher than that of the Caerin1.1 and Caerin1.9 peptide groups. When the peptide concentrations were increased to 40 μg / mL, the inhibitory effect of Caerin1.1 and Caerin1.9 peptides on the proliferation of prostate cancer cells was more significant, while TCMP-Y1 peptide had no significant inhibitory effect on the proliferation of DU145 cells. Calculate the IC50 values ​​of Caerin 1.1 peptide and Caerin 1.9 peptide. 50 The concentrations were 10.87 μg / mL and 14.50 μg / mL, respectively (Figure 1D). The results show that when the concentrations of Caerin1.1 and Caerin1.9 peptides were less than 5 μg / mL, their inhibitory effects on DU145 cells were not significant. However, as the concentrations of the two peptides increased sequentially, the inhibitory effects of Caerin1.1 and Caerin1.9 peptides on DU145 cells became increasingly apparent, and the higher the concentration, the lower the cell viability. With increasing concentrations of toad peptide TCMP-Y1, the proliferation of DU145 cells was not significantly inhibited, thus it can be inferred that TCMP-Y1 peptide has no inhibitory effect on the proliferation of DU145 cells.

[0069] Example 2 Plate Cloning Experiment

[0070] (1) Select DU145 cells with good growth status and cell density of 80% to 90% for plate colony formation experiment. After disinfection, rinsing, digestion, counting and centrifugation, DU145 cells were evenly seeded into two 6-well plates with a cell density of 800 cells / mL / well. The 6-well plates were gently shaken to ensure uniform cell distribution and then placed in an incubator for 24 hours.

[0071] (2) After 24 hours, the cells were observed to be well adhered to the culture medium under a microscope. Then, cell culture medium containing different concentrations of Caerin1.1 peptide and Caerin1.9 peptide was added to each group. The concentrations of Caerin1.1 group were 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL and 10 μg / mL, respectively; the concentrations of Caerin1.9 group were 0 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL and 12.5 μg / mL, respectively. After mixing well, the cells were incubated in an incubator for another 24 hours.

[0072] (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 cluster in the well with a drug concentration of 0 μg / mL is about 50 cells / cluster, stop the culture and culture for a total of 9 days.

[0073] (3) After terminating the culture, remove the culture medium, wash three times with pre-cooled PBS, and add 1 mL of 4% paraformaldehyde solution to each well for 30 min to fix.

[0074] (4) Remove the fixative, rinse 3 times, add 0.5 mL of crystal violet staining solution to each well, stain for 15 min; discard the crystal violet solution, rinse slowly with running water for about 15 min, and air dry naturally, then scan or photograph with a scanner.

[0075] The results of the plate colony formation experiment are shown in Figures 2 and 3. The plate colony formation experiment further demonstrated that Caerin 1.1 and Caerin 1.9 peptides could inhibit the proliferation of prostate cancer cells DU145. With increasing peptide concentration, the number of cells gradually decreased, indicating a decline in proliferative capacity. Compared with the control group (concentration 0 μg / mL), the difference in cell colony number was statistically significant (P < 0.05), consistent with the results of the MTT assay.

[0076] The experimental results from Examples 1 and 2 show that Caerin 1.1 and Caerin 1.9 peptides significantly inhibited the proliferation of prostate cancer cells (DU145 cells) in a concentration-dependent manner; while TCMP-Y1 peptide showed no inhibitory effect on the proliferation of DU145 prostate cancer cells, indicating that it does not possess antitumor activity. The plate colony formation assay further verified that Caerin 1.1 and Caerin 1.9 peptides significantly inhibited the growth of DU145 tumor cells in a concentration-dependent manner.

[0077] Example 3 131 Preparation and quality identification of I-labeled Caerin 1.1 peptide, Caerin 1.9 peptide and TCMP-Y1 peptide

[0078] Previous experiments have confirmed that 100 μL of Na (370 MBq / mL, 10 mCi / mL, i.e., a total volume of 1 mCi) 131 The highest labeling efficiency was achieved when solution I was mixed with 40 μL (1 mg / mL) of Caerin 1.1 or Caerin 1.9 peptides; therefore, this ratio was used for all subsequent labeling. 131 I-Caerin 1.1 and 131 I-Caerin 1.9. Furthermore, based on preliminary experiments, it was found that... 131 When labeling TCMP-Y1 peptide with I, 100 μL (370 MBq / mL, 10 mCi / mL, i.e., a total volume of 1 mCi) Na 131 The highest labeling efficiency was achieved when solution I was used to label 100 μL (1 mg / mL) of TCMP-Y1 peptide. The specific operating steps are as follows:

[0079] (1) Take 40 μL (1 mg / mL) of Caerin1.1 peptide, 40 μL (1 mg / mL) of Caerin1.9 peptide and 100 μL (1 mg / mL) of TCMP-Y1 peptide and place them in three 1.5 mL sterile EP tubes for later use.

[0080] (2) Dissolve 50 mg of chloramine-T trihydrate (Maclean, China) in 50 mL of PBS to a concentration of 1 mg / mL and store in the dark;

[0081] (3) Customized three Na 131 Solution I, with a volume of 100 μL, had a radioactivity concentration of 370 MBq / mL (10 mCi / mL) as measured by an activity meter; 100 μL of Na+ was added to each EP tube containing the polypeptide. 131 Solution I was then added, followed by 100 μL of chloramine-T solution with a concentration of 1 mg / mL.

[0082] (4) At room temperature, use a vortex mixer to mix and vibrate for 10 minutes to complete the process. 131 Experiments using I-labeled Caerin1.1, Caerin1.9, and TCMP-Y1.

[0083] (4) Using physiological saline (NS, Yangzhou Zhongbao Pharmaceutical Co., Ltd., China) as the developing solvent, the radioactivity was assessed by thin-layer paper chromatography using a gamma counter (Zhongjia Optoelectronics Co., Ltd., China). 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The labeling rate of the I-TCMP-Y1 peptide was determined. γ-counting curves were plotted using GraphPad Prism 9.0.0 software, and the labeling rate was obtained by calculating the area under the curve.

[0084] Labeling rate = 131 I-Caerin 1.1 or 131 I-Caerin 1.9 or 131 I-TCMP-Y1 radioactive peak area integral / total radioactive peak area integral × 100%.

[0085] Rf value = 131 I-Caerin 1.1 or 131 I-Caerin 1.9 or 131 The distance I-TCMP-Y1 moves / the distance the developing propellant front moves.

[0086] The results are shown in Figure 4. 131 I-Caerin 1.1 131 I-Caerin 1.9 131 The radiolabeling rates of I-TCMP-Y1 were (96.86% ± 0.54)%, (95.39% ± 0.50)%, and (94.92% ± 0.14)%, respectively, with all three having a labeling rate of around 95%.

[0087] Example 4 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 Determination of the lipid-water partition coefficient of I-TCMP-Y1

[0088] This embodiment demonstrates... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The lipid-water partition coefficient of I-TCMP-Y1 was determined to infer the excretion pathways of the three radiolabeled products in vivo. The procedure is as follows:

[0089] (1) Prepare 3 EP tubes, each filled with 500 μL of n-octanol (lipid phase) and 500 μL of physiological saline (aqueous phase);

[0090] (2) Prepared according to the above labeling method 131 I-Caerin 1.1 131 I-Caerin 1.9 or 131 I-TCMP-Y1, take 50μL from each into an EP tube, seal and mix well;

[0091] (3) Place the EP tube in a centrifuge, balance it, and centrifuge at 4000 rpm for 5 min. After centrifugation, the solution can be clearly divided into two phases: the upper phase is lipid phase, and the lower phase is aqueous phase.

[0092] (4) Take 100 μL of liquid from the upper lipid phase and the lower aqueous phase and place them in two glass counting tubes respectively, and measure their γ count using a γ radioimmunoassay counter;

[0093] (5) Calculate the lipid-water partition coefficient (log P), log P = log[(lipid phase γ count - background γ count) / (water phase γ count - background γ count)].

[0094] Table 1 131 CPM count and lipid-water partition coefficient of I-Caerin 1.1 in lipid and aqueous phases

[0095] Table 2 131 CPM count and lipid-water partition coefficient of I-Caerin 1.9 in lipid and aqueous phases

[0096] Table 3 131 CPM count and lipid-water partition coefficient of I-TCMP-Y1 in lipid and aqueous phases

[0097] 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The lipid-water partition coefficients of I-TCMP-Y1 are shown in Tables 1, 2, and 3, respectively. 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The 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, indicating that... 131 I-Caerin 1.1 and 131 I-Caerin 1.9 is weakly lipophilic, while 131 I-TCMP-Y1 is weakly water-soluble.

[0098] Example 5 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 Determination of the stability of I-TCMP-Y1

[0099] This embodiment will... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131I-TCMP-Y1 was placed in fetal bovine serum or physiological saline at 25°C or 37°C, and its radiochemical purity (RCP) was measured at different time points to assess its in vitro stability. The experiment was repeated three times, following these steps:

[0100] (1) Place 100 μL of [unspecified substance] into three clean 1.5 mL EP tubes respectively. 131 I-Caerin 1.1 131 I-Caerin 1.9 or 131 I-TCMP-Y1, and sealed and stored in a constant temperature water tank at 37℃ and room temperature (25℃);

[0101] (2) Take 20 μL from each of the labeled solutions and place them in 80 μL of fetal bovine serum or 80 μL of physiological saline. Mix them thoroughly using a vortex mixer and store at room temperature (25°C).

[0102] (3) Take 20 μL from each of the labeled solutions and place them in 80 μL of fetal bovine serum or 80 μL of physiological saline. Mix them thoroughly using a vortex mixer and store them in a constant temperature water bath at 37°C.

[0103] (4) The radiochemical purity of each mixture was determined by paper chromatography at different time periods (0h, 24h and 72h), and the experiment was repeated three times.

[0104] The results are shown in Figure 5. All three labeled products exhibited high radiochemical purity (RCP) after being placed in fetal bovine serum (FBS) or normal saline (NS) at room temperature (25°C), 37°C, and for different periods. (See Figures 5A, 5D, and 5G). 131 I-Caerin 1.1 131 I-Caerin 1.1 and fetal bovine serum mixture and 131 After a mixture of I-Caerin 1.1 and physiological saline was stored at 25°C for 72 hours, the radiochemical purities were (84.62±1.78)%, (85.19±1.49)%, and (84.81±0.79)%, respectively. The RCPs of the three compounds after being placed in a constant temperature water bath at 37°C for 72 hours were (84.41±0.79)%, (85.36±1.08)%, and (85.12±1.77)%, respectively. (See Figures 5B, 5E, and 5H.) 131 I-Caerin 1.9 131 I-Caerin 1.9 and fetal bovine serum mixture and 131After storage at 25°C for 72 hours, the radiochemical purities of I-Caerin 1.9 and physiological saline mixtures were (85.99±2.23)%, (85.87±1.14)%, and (85.82±2.17)%, respectively; the RCPs of the three mixtures after being placed in a constant temperature water bath at 37°C for 72 hours were (86.42±2.46)%, (87.54±0.79)%, and (87.26±1.10)%, respectively. As shown in Figures 5C, 5F, and 5I, the simple... 131 I-TCMP-Y1 sample, 131 I-TCMP-Y1 and fetal bovine serum mixture and 131 After I-TCMP-Y1 and physiological saline mixtures were stored at 25°C for 72 h, their radiochemical purities were (87.70±0.60)%, (87.48±0.82)%, and (87.78±1.19)%, respectively; after being placed in a constant temperature water bath at 37°C for 72 h, their radiochemical purities were (88.09±1.05)%, (87.71±0.74)%, and (85.94±1.43)%, respectively.

[0105] The above experimental results show that, regardless of 131 I-Caerin 1.1 131 I-Caerin 1.9 or 131 I-TCMP-Y1, the three labeled products, still exhibited high in vitro stability after being stored at different temperatures (25℃, 37℃) and in different solutions (fetal bovine serum or physiological saline) for 72 hours.

[0106] Based on the experimental results of Examples 3-5, it can be seen that the chloramine-T direct labeling method yields... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The labeling rate of I-TCMP-Y1 was around 95%. Preliminary inferences suggest... 131 I-Caerin 1.1 and 131 I-Caerin 1.9 is weakly lipid-soluble and may be excreted through the liver; 131 I-TCMP-Y1 is weakly water-soluble and may be excreted via the kidneys. Three labeled products... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 exhibits good in vitro stability in fetal bovine serum or saline at different temperatures (25℃, 37℃).

[0107] Example 6 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131Cellular uptake assay of I-TCMP-Y1

[0108] This embodiment examines the effects of DU145 cells on different time points. 131 I-Caerin 1.1 131 I-Caerin 1.9 131 I-TCMP-Y1 and Na 131 I uptake status, assessing DU145 cells' response to 131 I-Caerin 1.1 131 I-Caerin 1.9 131 I-TCMP-Y1 and Na 131 The binding ability of I operates as follows:

[0109] (1) Select DU145 cells in the logarithmic growth phase, digest them into a cell suspension (see 2.3.4 for specific steps on cell digestion), count the cells, and resuspend them to 1×10⁻⁶. 5 cells / mL;

[0110] (2) The cell suspension was evenly seeded into eight 24-well plates, with a cell count of 5 × 10⁶ cells. 4 500 μL / well, continue incubation for 24 h;

[0111] (3) After the cells adhered to the culture vessel, discard the supernatant, wash twice, add 0.5 mL of serum-free pure culture medium to each well, divide into 4 experimental groups, with 3 replicates and 1 positive control well in each group; the drug concentrations are as follows:

[0112] A: 131 I-Caerin 1.1 group: 259 KBq / 2 μL / well;

[0113] B: 131 I-Caerin 1.9 group: 259 KBq / 2 μL / well;

[0114] C: 131 Group I-TCMP-Y1: 259KBq / 2μL / well;

[0115] D:Na 131 Group I: 259KBq / 2μL / well;

[0116] (4) After adding the drug according to the above plan, place the 24-well plate in the incubator and incubate for 2, 4, 6 and 24 hours respectively; after the incubation, aspirate the supernatant and wash with fresh pre-cooled PBS, repeat twice (collect the supernatant and washing solution in the corresponding test tubes for the positive control wells), add 200 μL of trypsin to all wells for digestion, wash with 400 μL of fresh pre-cooled PBS each time, repeat 3 times and collect the washing solution;

[0117] (5) Measure the radioactivity count in each test tube and calculate the cell binding rate at different time points.

[0118] Cell binding rate = (average γ count in 3 replicates per group / γ count in the corresponding positive control wells per group) × 100%.

[0119] The results of the cell uptake experiment are shown in Figure 6A. In the cell uptake experiment, DU145 cells and 131 I-Caerin 1.1 131 I-Caerin 1.9 131 The binding rate of I-TCMP-Y1 increased with time, reaching its highest value at 24 hours, with binding rates of (19.60±0.81)%, (18.65±0.53)%, and (9.00±1.45)%, respectively. 131 I-Caerin 1.1 131 The increase of I-Caerin 1.9 compared to 131 I-TCMP-Y1 is faster; however, DU145 cells have virtually no ability to take up Na. 131 The binding rate of I was only (0.57±0.03)% after 24 hours.

[0120] Example 7 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 Cell elution assay of I-TCMP-Y1

[0121] (1) Select logarithmically growing DU145 cells, digest, count, centrifuge, and resuspend to 1×10⁶ cells. 5 cells / mL;

[0122] (2) The above cell suspension was seeded into 8 wells of 24-well plates, with a seeding volume of 500 μL per well, and cultured in an incubator for 24 h.

[0123] (3) After the cells adhered to the culture vessel, the supernatant was aspirated, and the cells were rinsed twice. 0.5 mL of serum-free culture medium was added to each well. The cells were divided into four experimental groups, with three replicates and one positive control well in each group. The drug concentrations were as follows:

[0124] A: 131 I-Caerin 1.1 group: 259 KBq / 2 μL / well;

[0125] B: 131 I-Caerin 1.9 group: 259 KBq / 2 μL / well;

[0126] C: 131 Group I-TCMP-Y1: 259KBq / 2μL / well;

[0127] D:Na 131 Group I: 259KBq / 2μL / well;

[0128] (4) Continue incubation in the incubator for 24 hours; after 24 hours, discard the supernatant, rinse twice, add 0.5 mL of serum-free culture medium to each well, and incubate 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 washing solution in the corresponding test tubes for the positive control wells), add 200 μL of trypsin to each well to digest the cells, wash with fresh pre-cooled PBS, repeat three times and collect the washing solution;

[0129] (5) Measure the γ count in each test tube and calculate the cell binding rate of the four groups at each time point. Cell binding rate = (average γ count of 3 replicates in each group / γ count of the corresponding positive control well in each group) × 100%.

[0130] The results of the cell elution experiment are shown in Figure 6B. 131 I-Caerin 1.1 131 I-Caerin 1.9 131 After incubation with I-TCMP-Y1 for 24 hours, followed by elution, all three drugs showed high binding rates to DU145 cells. 131 The binding rate of I-TCMP-Y1 to DU145 cells was slightly lower than that of the other two drug groups, and it plateaued after 24 hours. The binding rates after elution and reculturing for 24 hours were (61.64±1.07)%, (46.68±1.64)%, and (66.65±0.80)%, respectively. Meanwhile, the binding rates of DU145 cells to Na... 131 The binding rate of I was only (3.03±0.21)%.

[0131] The above cell uptake and elution experiments showed that all three radiolabeled products could be taken up by DU145 cells.

[0132] Example 8 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 cytotoxic proliferation assay

[0133] The CCK-8 assay was used to verify the activity of DU145 cells in response to Caerin 1.1 peptide, Caerin 1.9 peptide, TCMP-Y1 peptide, and Na+. 131 I, 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131The study investigated the differences in cell viability under I-TCMP-Y1 treatment and compared the inhibitory effects of different drugs on DU145 cell proliferation. The specific experimental procedures are as follows:

[0134] (1) Select DU145 cells in the logarithmic growth phase with a cell density of 80%–90%, digest, centrifuge, count, and resuspend to 5.0 × 10⁻⁶ cells. 4 Cells / mL: Add 100 μL of cell suspension to each well of a 96-well plate using a pipette, resulting in a cell density of 5 × 10⁶ cells / mL. 3 Cells per well, fill the edge wells of the 96-well plate with 100 μL of sterile PBS. After adding all the PBS, gently shake the plate to distribute the cells evenly and then incubate at 37°C and 5% CO2 for 24 hours.

[0135] (2) After 24 hours, when the cells had grown to 80% of the well, the drug was added. The drug concentrations for each group were as follows:

[0136] A: 131 I-Caerin 1.1 group: 2500 KBq / mL, 5000 KBq / mL, 10000 KBq / mL, 20000 KBq / mL; 131 I-Caerin1.9 group, 131 I-TCMP-Y1 group and Na 131 Group I 131 I-Caerin1.1 group;

[0137] B: Based on the amounts in group A, the corresponding Caerin 1.1 peptide group and Caerin 1.9 peptide group were 2.8 μg / mL, 5.6 μg / mL, 11.2 μg / mL, and 22.4 μg / mL, respectively, and the corresponding TCMP-Y1 peptide group were 7 μg / mL, 14 μg / mL, 28 μg / mL, and 56 μg / mL, respectively; each group was set up with 3 parallel replicates, and three wells without drug were used as control groups;

[0138] (3) Continue culturing for 24 hours, add 10 μL of CCK-8 reagent to each well, and culture in the dark for 4 hours. Use a full-wavelength microplate reader (wavelength 450 nm) to detect the OD value of each well and calculate the cell survival rate of each group.

[0139] Cell viability = (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%.

[0140] The experimental results are shown in Figure 7, specifically Figures 7A, 7B, and 7C. When 131 I-Caerin 1.1 131 When the radioactivity concentration of I-Caerin 1.9 reaches 20000 KBq / mL, 131I-Caerin1.1 group, 131 The survival rates of DU145 cells in the I-Caerin 1.9 group were (7.18±0.93)% and (19.58±1.80)%, respectively; while 131 The cell viability of the I-TCMP-Y1 group remained as high as (39.17±0.83)%. As shown in Figures 7D, 7E, and 7F, compared with the corresponding peptide-only groups at the same peptide concentration, 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 Cell viability in the I-TCMP-Y1 group was consistently low, and the differences were statistically significant (P < 0.05). Furthermore, this invention also found that Na... 131 I only showed an inhibitory effect on the proliferation of DU145 prostate cancer cells, at which point the cell survival rate was (70.09±3.39)%. As shown in Figures 7G, ​​7H, and 7I, at higher concentrations... 131 I-Caerin1.1 and 131 Between I-Caerin 1.9 131 I-Caerin1.1 and 131 Between I-TCMP-Y1 131 I-Caerin1.9 and 131 The comparison of the cytotoxic effects of I-TCMP-Y1 on DU145 cells showed statistically significant differences (P < 0.05). Similarly, at higher concentrations, Caerin1.1 peptide versus Caerin1.9 peptide, Caerin1.1 peptide versus TCMP-Y1 peptide, and Caerin1.9 peptide versus TCMP-Y1 peptide showed statistically significant differences in their inhibitory effects on DU145 cell proliferation (P < 0.05), which is consistent with previous MTT assay results. These results confirm that the CCK-8 assay validated... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 can inhibit the proliferation of DU145 cells. As the radioactive concentration increases, the cytotoxic effects of the three radiopharmaceuticals gradually increase, and the survival rate of DU145 cells gradually decreases.

[0141] Example 9: Establishment of a DU145 cell tumor-bearing nude mouse model

[0142] (1) Select DU145 cells in the logarithmic growth phase with a cell density of 80%–90%. After digestion, centrifugation, and counting, immediately resuspend the cells in pre-cooled sterile PBS and adjust the cell concentration to 1×10⁻⁶. 7Cells / mL, transfer the cell suspension to a 5mL EP tube and place it in an ice box;

[0143] (2) After disinfecting the skin under the right upper limb axilla of nude mice with alcohol disinfectant, 100 μL of the above-mentioned DU145 cell suspension was drawn with a syringe, and the cell count was 1 × 10⁻⁶. 6 One per mouse, slowly injected into the skin under the armpit; a raised, transparent blister can be seen under the right armpit of the nude mouse.

[0144] (3) After about two days, the blisters will be absorbed, and a rice-grain-sized growth will appear on the skin of the right armpit. Regularly observe the condition of the nude mice and the growth of the tumor, and change the feed and water with fresh water. Measure the size of the tumor with electronic calipers every two days. When the tumor size is close to 3-4 mm, it indicates that the nude mouse model of prostate cancer has been successfully established, and subsequent experiments can be carried out. Tumor volume = width × width × length / 2.

[0145] The results are shown in Figure 8. Figures 8A and 8B show that, prior to euthanasia, the PBS group, Caerin 1.1 group, Caerin 1.9 group, TCMP-Y1 group, and Na... 131 Group I, 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The tumor volume in group I-TCMP-Y1 was (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 pre-treatment levels, tumor volume increased in all groups, but the tumor volume increase was significantly greater in the three groups treated with iodine-131-labeled peptides compared to the PBS group. 131Groups I, Caerin 1.1, Caerin 1.9, and TCMP-Y1 showed slower progression. At the start of treatment, there was no statistically significant difference in tumor size among the groups (P > 0.05). As shown in Figure 8C, from the end of treatment until euthanasia, the body weights of the 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, respectively. It can be seen that the body weight of the mice in each group increased compared to before treatment. As shown in Figure 8D, after euthanizing the nude mice, they were dissected, tumor tissues were separated, and weighed. The tumor weights for 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. It can be seen that the separated... 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The tumor size in the I-TCMP-Y1 group was significantly smaller than that in other groups, proving that... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The I-TCMP-Y1 group also has the effect of inhibiting the growth of prostate cancer in vivo.

[0146] Example 10 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 In vivo therapeutic trials of I-TCMP-Y1

[0147] (1) Before the in vivo experiment, in order to block the thyroid gland and reduce thyroid uptake in nude mice. 131 I. Feed the nude mice used in the experiment with 0.1% potassium iodide 3 days in advance;

[0148] (2) The successfully modeled DU145 tumor-bearing nude mice were randomly divided into 8 groups, with 4 mice in each group: Caerin 1.1 peptide group, Caerin 1.9 peptide group, TCMP-Y1 peptide group, PBS group (control group), and PBS group (control group). 131 I-Caerin 1.1 group, 131 I-Caerin1.9 group, 131 I-TCMP-Y1 group, Na- 131In Group I, each nude mouse received 100 μL of drug per treatment. The Caerin 1.1 and Caerin 1.9 peptide groups were administered 100 μL of PBS solution containing either 8 μg Caerin 1.1 or 8 μg Caerin 1.9 peptide; the TCMP-Y1 peptide group was administered 100 μL of PBS solution containing 20 μg TCMP-Y1 peptide; the PBS group (control group) was administered 100 μL of sterile PBS solution; Na… 131 Group I was given sterile Na containing 200 μCi (7.4 MBq). 131 Solution I; 131 I-Caerin 1.1 group and 131 The I-Caerin1.9 group was given either 8 μg Caerin1.1 peptide or 8 μg Caerin1.9 peptide and 200 μCi Na 131 A mixture of I; 131 The I-TCMP-Y1 group was given 20 μg of TCMP-Y1 peptide and 200 μCi Na 131 A mixture of I;

[0149] (3) Inject the drug into the tumor once every 2 days, for a total of 3 injections. Before each injection, measure the length and width of the tumor in the nude mouse with electronic calipers and calculate the volume. Weigh the nude mouse with an electronic balance. On the 5th day after the last treatment, euthanize the nude mouse by cervical dislocation, dissect the tumor and weigh it.

[0150] The experimental results are shown in Figure 9. Tumor tissue specimens isolated from different treatment groups after euthanasia of nude mice show... 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The tumor volume in the I-TCMP-Y1 group was generally smaller, indicating that 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 both have anti-prostate cancer activity.

[0151] Figure 10 shows the results of H&E staining of tumor sections from different treatment groups and the statistical graph of differences in the area of ​​cell degeneration and necrosis among different treatment groups. In the PBS group, tumor cells were densely packed with large, deeply stained nuclei, a proliferative change, and a few cells showed degeneration and necrosis. In the Caerin1.1 group, Caerin1.9 group, and TCMP-Y1 group, a small amount of cell degeneration and necrosis were observed, and there was no statistically significant difference compared with the PBS control group (P>0.05). 131 I-Caerin 1.1 131 I-Caerin 1.9 and131 All three groups of tumor cells (I-TCMP-Y1) showed significant degeneration and necrosis, exhibiting homogeneous red staining and disrupted cell structure (Figure 10A). 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The I-TCMP-Y1 group was compared with the PBS group and Na... 131 In Group I, the area of ​​tumor necrosis showed a significant difference (P < 0.05). 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The I-TCMP-Y1 group showed a significant difference in tumor necrosis area compared to their respective peptide groups (P < 0.05) (Figure 10B). This result indicates that... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 can kill tumor cells and inhibit tumor proliferation in vivo.

[0152] Based on the experimental results of Examples 6 to 10, it can be seen that 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 can be taken up by prostate cancer (DU145) cells, and 131 I-Caerin 1.1 131 I-Caerin 1.9 can be stably retained in DU145 cells. 131 The ability of I-TCMP-Y1 to remain in DU145 cells is relatively... 131 I-Caerin 1.1 131 I-Caerin 1.9 was weak; while DU145 cells hardly took up any. 131 I. 131 I-Caerin 1.1 131 I-Caerin 1.9 or 131 I-TCMP-Y1 showed significant inhibitory and killing effects on the proliferation of DU145 cells; and compared with Caerin 1.1 peptide and Caerin 1.9 peptide at the same concentration, 131 I-Caerin 1.1 131 I-Caerin 1.9 showed stronger inhibitory activity, while TCMP-Y1 had no significant inhibitory effect on DU145 cells. In in vivo therapeutic experiments, compared with the control group, the peptide-only group, and the iodine-131-only group, tumor volume and tumor weight were observed. 131 I-Caerin 1.1131 I-Caerin 1.9 and 131 I-TCMP-Y1 significantly inhibited the growth of prostate cancer. Until the nude mice were euthanized, the body weight of each group of mice increased compared to before treatment, which may indicate... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 exhibits good biocompatibility.

[0153] The MTT assay results of this invention show that Caerin1.1 and Caerin1.9 peptides can inhibit the proliferation of prostate cancer cells (DU145) in vitro in a concentration-dependent manner, with IC50 values ​​of 10.87 μg / mL and 14.50 μg / mL for Caerin1.1 and Caerin1.9, respectively. In contrast, TCMP-Y1 peptide showed no significant inhibitory effect on DU145 cell proliferation. The results of the plate colony assay were similar to those of the MTT assay, demonstrating that the cytotoxic effect of the drugs on DU145 cell proliferation became more significant with increasing concentrations of Caerin1.1 and Caerin1.9 peptides.

[0154] This invention uses the chloramine-T method to prepare three types of... 131 I-labeled products, i.e. 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1, and the labeling rates of all three were around 95%. Experiments have shown that... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 maintains high stability in both NS and FBS at room temperature (25°C) and body temperature (37°C). This stability was demonstrated by measurements... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The lipid-water partition coefficient of I-TCMP-Y1, as known in this invention. 131 I-Caerin 1.1 131 The LogP values ​​for I-Caerin 1.9 were both >0, indicating that both drugs are weakly lipophilic. 131 LogP of I-TCMP-Y1 is all < 0, indicating that 131 I-TCMP-Y1 is weakly water-soluble, which proves... 131 I-Caerin 1.1 131 I-Caerin 1.9 is likely excreted primarily via the liver; while 131I-TCMP-Y1 is likely primarily excreted through the urinary system, potentially reducing the risk of radiation-induced liver injury. In cell uptake and elution experiments, with prolonged drug exposure... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 The binding rate and binding ability of I-TCMP-Y1 in prostate cancer cells DU145 gradually increased. 131 I-TCMP-Y1 is weaker than the former two, but both are higher than Na. 131 Group I, which indicates 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 can be taken up and stably retained by prostate cancer cells (DU145). This is because prostate cancer cells (DU145) cannot take up sodium. 131 The reason for this may be related to the fact that prostate cancer cells do not express the sodium-iodine transporter (NIS). In cell elution experiments, elution was performed after 2 hours, 4 hours, 6 hours, and 24 hours. 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 exhibits high retention capacity in prostate cancer cells (DU145), and 131 I-Caerin 1.1 131 I-Caerin 1.9 ratio 131 I-TCMP-Y1 has a stronger binding force.

[0155] The CCK8 experimental results confirm that... 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 exhibits significant proliferative toxicity against prostate cancer cells (DU145), and is more potent than Na+ alone. 131 I. Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide all showed stronger effects, while the single peptide and TCMP-Y1 peptide had no significant toxic effect on DU145 cells. This may be because Caerin 1.1 peptide, Caerin 1.9 peptide, and TCMP-Y1 peptide can... 131 This is related to I being carried into the cell, which is consistent with the results of the previous cell uptake and cell elution experiments.

[0156] In the in vivo experimental portion, this invention chose intratumoral injection as the treatment method because intratumoral administration has the advantage of avoiding the problems associated with systemic administration, such as poor tumor penetration, excessively rapid drug clearance, and damage to healthy tissue. Compared with the control group (PBS group), the simple peptide groups (Caerin 1.1 group, Caerin 1.9 group, TCMP-Y1 group), and Na... 131 Compared to Group I, 131 I-Caerin1.1 group, 131 I-Caerin 1.9 group and 131 The I-TCMP-Y1 group showed lower tumor volume growth rate and tumor weight, with statistically significant differences (P < 0.05); however, there was no statistically significant difference in tumor volume growth rate among the three groups treated with iodine-labeled peptides (P > 0.05); regarding tumor weight, 131 I-Caerin 1.1 group and 131 Between I-Caerin 1.9 groups 131 I-Caerin 1.9 group and 131 There was no statistically significant difference between the I-TCMP-Y1 groups (P > 0.05), while 131 I-Caerin 1.1 group and 131 There was a statistically significant difference between the I-TCMP-Y1 groups (P < 0.05).

[0157] Through cytotoxicity proliferation assays and in vivo experiments, this invention found that compared with Caerin1.1, Caerin1.9, and TCMP-Y1, 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 exhibits a stronger tumor-suppressive effect against prostate cancer. Therefore, this invention hypothesizes that all three peptides carry [tumor-suppressive activity]. 131 I. The ability to enter tumor cells. Entering the cell... 131 I-Caerin 1.1 and 131 I-Caerin 1.9 possesses both the biological radiation effects of radionuclides and the anticancer effects of small molecule peptides, thus its biological effects are superior to those of Na. 131 I, Caerin 1.1 and Caerin 1.9; Based on the previous MTT assay, it was found that the TCMP-Y1 peptide alone had no significant inhibitory effect on the proliferation of DU145 cells. Therefore, this invention infers... 131 The tumor-suppressive effect of I-TCMP-Y1 may be due to 131 The radiobiological effects of I.

[0158] In summary, the experimental results of this invention show that the TCMP-Y1 polypeptide alone has no significant inhibitory effect on the proliferation of prostate cancer cells DU145; while Caerin 1.1 and Caerin 1.9 alone have an inhibitory effect on the proliferation of prostate cancer cells DU145. 131 I-Caerin 1.1 131 I-Caerin 1.9 and 131 I-TCMP-Y1 can be taken up and stably retained by prostate cancer cells DU145, and has a more significant inhibitory effect on tumor growth. It is also water-soluble. 131 I-TCMP-Y1 is primarily excreted through the kidneys and urinary system, thus reducing the risk of radiation-induced liver injury. This indicates that TCMP-Y1 is a small molecule peptide that effectively introduces radionuclides into the body, improving drug safety and tumor targeting, and could serve as a potential drug for intratumoral radiation therapy.

Claims

1. A small molecule polypeptide TCMP-Y1, characterized in that, Its amino acid sequence is YFKAWGWPIDDATTEHL-NH2.

2. 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 radiolabeled iodine-131 is bound to its amino acid groups.

3. The method for preparing the iodine-131-labeled small molecule polypeptide TCMP-Y1 according to claim 2, characterized in that, To employ the chloramine-T method, a solution of small molecule polypeptide TCMP-Y1 and Na- 131 Solution I and chloramine-T solution were mixed by shaking at room temperature, and then separated by chromatography. 131 The small molecule polypeptide TCMP-Y1 labeled with I is obtained.

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

5. The use of the iodine-131 labeled small molecule polypeptide TCMP-Y1 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 prostate cancer.

7. A tumor treatment drug, characterized in that, The small molecule polypeptide TCMP-Y1 labeled with iodine-131 as described in claim 2 is contained in the present invention.

8. The drug according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

9. The use of Caerin1.1 polypeptide or iodine-131-labeled Caerin1.1 polypeptide in the preparation of drugs 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 has radioactively labeled iodine-131 bound to the amino acid groups of the Caerin1.1 polypeptide.

10. The use of Caerin 1.9 polypeptide or iodine-131-labeled Caerin 1.9 polypeptide in the preparation of drugs 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 has radioactively labeled iodine-131 bound to the amino acid group of the Caerin1.9 polypeptide.

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