A novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase and a preparation method and application thereof

Abstract

The application discloses a novel radionuclide therapeutic drug targeting nucleolus DDX24 helicase and a preparation method and application thereof. 64 The Cu-DOTA-TDP-2 has good tumor targeting uptake specificity, and is expected to provide a precise molecular imaging method for esophageal squamous cell carcinoma and other DDX24 high-expression malignant tumors; the novel radionuclide targeted drug 177 The Lu-DOTA-TDP-2 has high radiochemical yield and specific activity, good chemical stability, high affinity for DDX24 helicase, and a simple and easy-to-operate preparation method, and can be used for the treatment of DDX24 high-expression malignant tumors; the novel radionuclide targeted drug 177 The Lu-DOTA-TDP-2 has excellent biological safety performance and good tumor inhibition effect in esophageal squamous cell carcinoma and other DDX24 high-expression malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a novel radionuclide therapeutic drug that targets nucleolar DDX24 helicase, its preparation method, and its application. Background Technology

[0002] In recent years, although the efficacy of esophageal squamous cell carcinoma has improved to some extent through comprehensive treatment including surgery, radiotherapy, and systemic drugs, its prognosis remains poor, with more than 70% of patients dying within 5 years of treatment. Therefore, finding new therapeutic targets and developing new treatment methods are of great clinical significance for improving the prognosis of patients with esophageal squamous cell carcinoma.

[0003] Targeted radionuclide therapy utilizes molecular carriers with specific high affinity for tumor cells to deliver radionuclides directly into tumor tissue, where they bind to specific receptors. The radionuclides then decay, generating radiation that kills tumor cells at close range. Localized radiation therapy to the lesion site can alter cell state, thereby alleviating pain symptoms caused by tumors and other diseases. It can be used to treat various diseases such as hyperthyroidism and psoriasis, offering a wide range of therapeutic applications. This treatment largely preserves the integrity of organs and tissues and can cause devastating damage to cancer cells in their growth and development process, thus achieving good therapeutic effects. However, the treatment requires destroying or killing tumor cells to achieve its effect, which may significantly impact bone marrow, damaging the hematopoietic system and leading to insufficient blood supply, potentially causing anemia. Radionuclide therapy may also cause significant damage to gastrointestinal function, leading to adverse reactions such as nausea and vomiting. Furthermore, residual tumor cells may remain in adjacent tissues, causing disease recurrence, resulting in limited treatment effectiveness and a high recurrence rate.

[0004] The advantages of radionuclide therapy mainly include its wide applicability and good therapeutic effect, while its disadvantages include limited therapeutic effect and significant adverse reactions. Radionuclide therapy refers to the treatment of various tissues and organs in the human body using the radioactive properties of radionuclides, achieving the effects and efficacy of irradiation and radiotherapy. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a novel radionuclide therapeutic drug that targets nucleolar DDX24 helicase.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned radionuclide therapeutic drug.

[0007] Another object of the present invention is to provide the application of the above-mentioned radionuclide therapeutic drug.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase is obtained by combining a radionuclide with a polypeptide that targets nucleolar DDX24 helicase.

[0010] The polypeptide targeting nucleolar DDX24 helicase is a polypeptide containing the amino acid residue TVCTWYCYCAAYQRA; preferably, polypeptide TDP-2.

[0011] The amino acid sequence of the polypeptide TDP-2 is TVCTWYCYCAAYQRARRRRRRRR.

[0012] The radioactive nuclide mentioned is a radioactive nuclide capable of undergoing β-ray decay or α-ray decay.

[0013] The radionuclides capable of undergoing β-ray decay include 177 Lu、 203 Hg, 191 Os、 198 Au、 137 Cs、 133 Xe, 132 I, 131 I, 130 I, 125 I, 123 I, 132 Te、 99 Te、 99 Mo、 91 Y、 90 Y、 90 Sr、 89 Sr、 87 Rb、 86 Rb、 85 Kr、 82 Br、 67 Cu、 60 Co、 59 Fe、 49 Ca, 47 Ca, 45 Ca, 43 K, 38 Cl、 33 P, 32 P, 24 Na、 14 C, or 3 At least one of H; preferably 64 Cu or 177 Lu.

[0014] The radionuclides capable of undergoing alpha-ray decay include222 Rn、 226 Ra、 228 Th、 238 Pu, or 241 At least one of Am.

[0015] The radionuclide therapeutic drug is bound by a metal chelating agent; preferably, the metal chelating agent is DOTA.

[0016] A method for preparing a novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase includes the following steps:

[0017] (1) Preparation of a polypeptide precursor targeting DOTA-targeting nucleolar DDX24 helicase;

[0018] (2) Add the DOTA-targeting nucleolar DDX24 helicase polypeptide precursor to the buffer solution, then add the radionuclide, adjust the pH, and react to obtain a novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase.

[0019] The amount of radionuclide added is 10-20 mCi of radionuclide per mg of DOTA-targeting nucleolar DDX24 helicase polypeptide precursor.

[0020] The buffer solution is sodium acetate buffer.

[0021] The pH adjustment mentioned refers to adjusting the pH to 4-6.

[0022] The reaction temperature is 35–50°C.

[0023] The reaction time is 10–80 min.

[0024] The method for synthesizing the polypeptide precursor of DOTA-targeting nucleolar DDX24 helicase includes the following steps:

[0025] S1. Weigh 0.11g (0.05mmol) of Fmoc-Arg(Pbf)-Wang Resin, place it in a reactor, and add an appropriate amount of DMF to soak for 2 hours;

[0026] S2. Drain the DMF, add an appropriate amount of 20% Pip / DMF, deprotect under nitrogen for 0.5 hours, wash the DMF 5 times, and the ninhydrin test will show a deep blue color.

[0027] S3. Add raw materials according to the equivalent ratio (AA:DIC:HoBt=3:3:3), add an appropriate amount of DMF, react under nitrogen until the ninhydrin test is clear, and wash with DMF 3 times after drying.

[0028] S4. Repeat steps 2 and 3, and after drying, wash twice with DMF, DCM and MeOH in sequence to obtain Compound1.

[0029] S5. Drain the resin in the reactor, transfer it to the cutting tube, add E solution (TFA:EDT:H2O:phenol:TIS = 90:2.5:2.5:2.5:2.5), and shake in a shaker for 2.5 hours.

[0030] S6. Filter the filtrate and collect it in a centrifuge tube. Add it to 6 times the volume of methyl tert-butyl ether and centrifuge to collect the solid. Wash the precipitated crude product with methyl tert-butyl ether 3 times to obtain 50 mg of the final crude product with a purity of 30%.

[0031] S7. Place the crude product in a drying pot and vacuum dry overnight. After purification, the precursor DOTA-TDP-2 is obtained.

[0032] The AA is a polypeptide that targets nucleolar DDX24 helicase.

[0033] The above-mentioned novel radionuclide therapeutic drugs targeting nucleolar DDX24 helicase are used in the preparation of antitumor drugs.

[0034] The drug contains one or more pharmaceutically acceptable carriers or excipients.

[0035] The excipients are slow-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorbent carriers, surfactants, or lubricants.

[0036] The aforementioned novel radionuclide therapeutic agents targeting nucleolar DDX24 helicase are used in tumor imaging and research for non-therapeutic and diagnostic purposes.

[0037] The tumors mentioned include at least one of the following: esophageal squamous cell carcinoma, liver cancer, gastric cancer, thyroid cancer, head and neck squamous cell carcinoma, pancreatic cancer, lymphoma, skin melanoma, lung cancer, colorectal cancer, gallbladder cancer, prostate cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, nasopharyngeal carcinoma, and bone cancer.

[0038] The present invention has the following advantages over the prior art:

[0039] (1) This invention first uses positron-emitting nuclides 64 A novel radionuclide probe was prepared by Cu-labeling targeting the DDX24 helicase DOTA-TDP-2. 64 Cu-DOTA-TDP-2 is used for non-invasive imaging of malignant tumors with high DDX24 expression. This invention utilizes... 64The method for labeling the peptide TDP-2 with Cu includes the following steps: 1 mg of the precursor DOTA-TDP-2 is added to 500 μl of sodium acetate buffer, followed by 0.8 mL of... 64 The CuCl2 (10.5 mCi) solution was adjusted to pH 5.0, and the reaction temperature was set to 45℃ for 15 min. The labeling rate was measured by radio-TLC. After labeling, the pH was adjusted to 7.0, and finally the solution was filtered through a microporous membrane for imaging.

[0040] (2) In order to investigate whether the DDX24 helicase-targeting polypeptide TDP-2 can be specifically taken up by tumors, this invention synthesized a novel radionuclide probe. 64 Cu-DOTA-TDP-2 was used in esophageal squamous cell carcinoma-bearing mice for tumor PET imaging. The results showed... 64 Cu-DOTA-TDP-2 exhibits specificity for tumor-targeted uptake.

[0041] (3) In order to study 177 The antitumor effect of Lu-DOTA-TDP-2 was demonstrated in this invention by constructing an esophageal squamous cell carcinoma-bearing mouse model and administering PBS via tail vein injection at doses of 5 MBq / 10 MBq / 15 MBq. 177 Lu-DOTA-TDP-2 was administered at a volume of 125 μl per mouse. Treatment was repeated 6 days after the first tail vein injection. The mice were observed for 36 consecutive days, starting from the day of the first treatment, monitoring tumor growth, survival, and liver and kidney function. Efficacy evaluation results showed that, compared to the PBS group, 177 Lu-DOTA-TDP-2 significantly inhibited the growth of subcutaneous esophageal squamous cell carcinoma and significantly prolonged the survival of tumor-bearing mice. Furthermore, 177 The dosage of Lu-DOTA-TDP-2 injection was positively correlated with tumor suppression, specifically, the tumors in the 15MBq group showed the slowest growth. Liver and kidney function indicators were repeatedly tested during treatment and showed no significant abnormalities.

[0042] The present invention has the following advantages over the prior art:

[0043] (1) The novel nuclide probe described in this invention 64 Cu-DOTA-TDP-2 exhibits good tumor-targeted uptake specificity and is expected to provide a precise molecular imaging method for malignant tumors with high DDX24 expression, such as esophageal squamous cell carcinoma.

[0044] (2) The novel radionuclide-targeted drug of the present invention 177 Lu-DOTA-TDP-2 exhibits high radiochemical yield and specific activity, good chemical stability, high affinity for DDX24 helicase, and a simple and easy preparation method, making it suitable for the treatment of malignant tumors with high DDX24 expression.

[0045] (3) The novel radionuclide-targeted drug of the present invention 177 Lu-DOTA-TDP-2 exhibits excellent biosafety and demonstrates good tumor suppression effects in malignant tumors with high DDX24 expression, such as esophageal squamous cell carcinoma, providing a new approach for the treatment of malignant tumors with high DDX24 expression. Attached Figure Description

[0046] Figure 1 yes 64 A schematic diagram and structural diagram of the preparation method of the Cu-DOTA-TDP-2 probe.

[0047] Figure 2 yes 64 Quality control results from the Cu-DOTA-TDP-2 high-performance liquid chromatography (HPLC) system. (A) 64 Typical radiometric HPLC chromatogram of Cu-DOTA-TDP-2. (B) Typical UV peak of DOTA-TDP-2 at 220 nm. (C) 64 Radio-HPLC of CuCl2.

[0048] Figure 3 This is an experimental verification of esophageal squamous cell carcinoma cell uptake and blockade. 64 The Cu-DOTA-TDP-2 probe targets and binds to DDX24.

[0049] Figure 4 yes 64 Typical images and quantitative analysis of Cu-DOTA-TDP-2micro PET / CT imaging. (A) Injection 64 Typical micro PET / CT images of mice bearing esophageal squamous cell carcinoma 1h to 45h after Cu-DOTA-TDP-2 probe injection (n=3); (B) Injection 64 Typical micro PET / CT images of esophageal squamous cell carcinoma-bearing mice after TDP-2 target blockade with Cu-DOTA-TDP-2 probe 2 hours prior; (C) 64 Time-activity curves of Cu-DOTA-TDP-2 probe injected 1h to 45h in tumor, tumor / muscle ratio, liver, kidney, lung, and muscle.

[0050] Figure 5 It is an injection 64 (A) Typical micro PET images of ex vivo organs and (B) Biodistribution results of ex vivo organs after 6 hours using the Cu-DOTA-TDP-2 probe.

[0051] Figure 6 It is a novel radionuclide-targeted drug 177The principle behind Lu-DOTA-TDP-2's targeted binding to the DDX24 helicase in esophageal squamous cell carcinoma cells.

[0052] Figure 7 It is a radionuclide-targeted drug 177 The labeling synthesis process of Lu-DOTA-TDP-2.

[0053] Figure 8 It is a representation 177 Binding capacity of Lu-DOTA-TDP-2. (A) In a competitive inhibition cell binding assay, unlabeled DOTA-TDP-2 competitively inhibited uptake in a dose-dependent manner. (B) Cell saturation binding assay. 177 The Kd value of Lu-DOTA-TDP-2.

[0054] Figure 9 The results were obtained by developing various radioactive materials with 0.01M EDTA solution on radio-TLC. (A) The detected substances are... 177 Lu 3+ The red peak represents radioactive free radicals. 177 Lu 3+ (B) The green radioactive single peak represents the labeled product. 177 The Lu-DOTA-TDP-2 (C) labeled product, after being left at room temperature for 144 hours, still exhibits a single radioactive peak at the origin.

[0055] Figure 10 This is a cell uptake assay analyzing esophageal squamous cell carcinoma cell line EC109 and radionuclides. 177 Lu and 177 Different Lu-labeled peptides ( 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 Differences in cell uptake after incubation of Lu-DOTA-Mdm2 / Mdmx-p53 for (A) 12 hours and (B) 24 hours.

[0056] Figure 11 This is a cell blockade experiment to analyze the interaction of esophageal squamous cell carcinoma EC109 cells with radionuclide after premature TDP-2 blockade. 177 Lu and 177 Different Lu-labeled peptides ( 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177Lu-DOTA-p53-2 and 177 Cell uptake after incubation of Lu-DOTA-Mdm2 / Mdmx-p53 for (A) 12 hours and (B) 24 hours.

[0057] Figure 12 After TDP-2 blockade, the comparison 177 Lu、 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 The inhibition of proliferation of esophageal squamous cell carcinoma EC109 cells treated with Lu-DOTA-Mdm2 / Mdmx-p53.

[0058] Figure 13 (A) Tail vein injection of PBS and different doses 177 (A) Schematic diagram of Lu-DOTA-TDP-2 treatment of esophageal squamous cell carcinoma-bearing mice; (B) Typical photographs of tumor-bearing mice in different treatment groups on day 36; (C) Comparison of tumor volume in different treatment groups on day 36, with statistically significant differences between the high-dose group and other groups; Esophageal squamous cell carcinoma-bearing mice injected with PBS via tail vein and different doses 177 (D) Subcutaneous tumor growth curve and (E) survival curve of tumor-bearing mice after Lu-DOTA-TDP-2 treatment.

[0059] Figure 14 It is (A) immunohistochemistry and (B) immunofluorescence detection of PBS and high dose 177 Ki67 expression in tumor tissue after Lu-DOTA-TDP-2 treatment.

[0060] Figure 15 It is (A) immunohistochemistry and (B) immunofluorescence detection of PBS and high dose 177 Expression of Caspase 3 in tumor tissue after Lu-DOTA-TDP-2 treatment.

[0061] Figure 16 It involves tail vein injection of PBS, in low, medium, and high doses. 177 Liver and kidney function test results on days 6 (D6), 12 (D12), and 36 (D36) after Lu-DOTA-TDP-2 treatment. ALT (alanine aminotransferase); AST (aspartate aminotransferase); BUN (blood urea nitrogen); Crea (blood creatinine). Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0063] Unless otherwise specified, the experimental conditions in the following implementation plan are generally based on standard experimental conditions or the experimental conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0064] Example 1

[0065] This embodiment involves a novel radionuclide probe. 64 Cu-DOTA-TDP-2, where 64 Cu is an isotope of copper with a half-life of 12.7 h; DOTA represents 1,4,7,10-tetraazacyclododecane-N,N',N",N"'tetraacetic acid, which is suitable for chelation. 64 A metal chelator of Cu; TDP-2, with the amino acid sequence Thr-Val-Cys-Thr-Trp-Tyr-Cys-Tyr-Cys-Ala-Ala-Tyr-Gln-Arg-Ala-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg, is a polypeptide probe capable of crossing the cell membrane barrier and specifically binding to the nucleolar protein DDX24 helicase.

[0066] 64 Labeling and synthesis of Cu-DOTA-TDP-2:

[0067] Add 1 mg of the precursor DOTA-TDP-2 (synthesized by Zhejiang Ontop Biotechnology Co., Ltd.) to 500 μl of sodium acetate buffer, then add 0.8 mL of [the solution is missing here]. 64 A CuCl2 solution (10.5 mCi, purchased from Guangzhou Atomic High-Tech Co., Ltd.) was prepared, the pH was adjusted to 5.0, and the reaction temperature was set to 45℃. After standing for 15 minutes, the product was obtained, with the following structural formula: Figure 1 As shown.

[0068] The synthesis method of the precursor DOTA-TDP-2 can be referred to the following steps:

[0069] S1. Weigh 0.11 g (0.05 mmol) of Fmoc-Arg(Pbf)-Wang Resin with a degree of substitution of 0.442 mmol / g, place it in a reactor, and add an appropriate amount of DMF to soak for 2 hours;

[0070] S2. Drain the DMF, add an appropriate amount of 20% Pip / DMF, deprotect under nitrogen for 0.5 hours, wash the DMF 5 times, and the ninhydrin test will show a deep blue color.

[0071] S3. Add raw materials according to the equivalent ratio (AA:DIC:HoBt=3:3:3), add an appropriate amount of DMF, react under nitrogen until the ninhydrin test is clear, and wash with DMF 3 times after drying.

[0072] S4. Repeat steps 2 and 3, and after drying, wash twice with DMF, DCM and MeOH in sequence to obtain Compound1.

[0073] S5. Drain the resin in the reactor, transfer it to the cutting tube, add E solution (TFA:EDT:H2O:phenol:TIS = 90:2.5:2.5:2.5:2.5), and shake in a shaker for 2.5 hours.

[0074] S6. Filter the filtrate and collect it in a centrifuge tube. Add it to 6 times the volume of methyl tert-butyl ether and centrifuge to collect the solid. Wash the precipitated crude product with methyl tert-butyl ether 3 times to obtain 50 mg of the final crude product with a purity of 30%.

[0075] S7. Place the crude product in a drying pot and vacuum dry overnight. After purification, the precursor DOTA-TDP-2 is obtained.

[0076] 64 Characterization of Cu-DOTA-TDP-2:

[0077] Labeled products 64 Quality control of Cu-DOTA-TDP-2 showed a single radioactive peak in analytical high-performance liquid chromatography, which was confirmed as a labeled product by comparison with its non-radioactive precursor DOTA-TDP-2. 64 The elution time of Cu-DOTA-TDP-2 was 10.49 minutes (e.g., Figure 2 As shown in A), the peak time for DOTA-TDP-2 is 11.38 minutes (as shown in A). Figure 2 (As shown in B), free 64 The elution time of Cu was 2.69 minutes (e.g., Figure 2 (as shown in C), 64 The radiochemical purity of Cu-DOTA-TDP-2 is >99%, and the radiochemical yield is >95%.

[0078] Example 2

[0079] one, 64 Cellular uptake and blocking assay of Cu-DOTA-TDP-2 probe:

[0080] This embodiment included cell uptake and blocking experiments to validate the novel molecular probe. 64Whether Cu-DOTA-TDP-2 can be specifically taken up by esophageal squamous cell carcinoma cells was investigated. EC109 cells in logarithmic growth phase were seeded at 5000 cells / well in a 96-well plate with 5 replicates. Each well contained 100 μL of Cu-DOTA-TDP-2. After 24 hours of incubation, 5 μCi was added to each well. 64 The Cu-DOTA-TDP-2 probe was incubated for 2 hours, 6 hours, and 12 hours, respectively. The results showed that the probe uptake rates in the three groups of cells were 25.5%, 27.1%, and 23.5%, respectively. In the blocking experiment, the cell plating method and number were exactly the same as in the cell uptake experiment. After 24 hours of plating, 5 μCi was added to each well. 64 The Cu-DOTA-TDP-2 probe was used, and 100 μM of TDP-2 peptide was added 2 hours before the detection to induce a competitive binding reaction, using the set detection time points as the standard. The remaining experimental procedures and measurement methods were the same as in the cell uptake experiment. The results showed that the probe uptake in EC109 cells decreased to 15.8% (p<0.01), 17.7% (p<0.0001), and 16.9% (p<0.01), respectively. Figure 3 As shown), this proves that 64 Cu-DOTA-TDP-2 has the ability to specifically target and bind to esophageal squamous cell carcinoma cells.

[0081] two, 64 Cu-DOTA-TDP-2 in vivo micro PET / CT imaging:

[0082] To verify at the living level 64 To assess the targeting ability of the Cu-DOTA-TDP-2 probe, this invention constructed an esophageal squamous cell carcinoma-bearing mouse model and evaluated, at the in vivo level, whether the probe could be specifically taken up by tumor tissue in these mice. Five- to six-week-old male Balb / c nude mice (purchased from Guangzhou Yancheng Biotechnology Co., Ltd.) were randomly divided into a non-blocking group and a blocking group (6 mice per group). The right hind leg of each mouse was disinfected with 75% alcohol, and 100 μl of esophageal squamous cell carcinoma EC109 cell suspension (approximately 5 × 10⁶ cells) was subcutaneously injected. 6 Tumor growth was observed every two days, with the size of the subcutaneous tumor measured and recorded using calipers. Micro PET / CT imaging was performed when the long diameter of the tumor increased to approximately 10–15 mm. Then, micro PET / CT imaging was performed 2h, 6h, 12h, 24h, 36h, and 45h after a probe was injected via the tail vein (injection dose: calculated at 20 μCi / g based on the tumor-bearing mouse's body weight). Results showed that the peak tumor uptake occurred 6h after probe injection, followed by a gradual decrease in tumor signal. However, a relatively strong tumor uptake signal was still visible 24h after probe injection (e.g., [missing information - likely a typo]). Figure 4As shown in Figure A), this demonstrates that the probe can be effectively taken up by the tumor and exhibits a good retention effect in tumor tissue. Furthermore, to demonstrate the targeted binding in esophageal squamous cell carcinoma... 64 To further investigate the specificity of Cu-DOTA-TDP-2, we conducted an inhibition experiment. The results showed that after intraperitoneal injection of TDP-2 for inhibition, the tumor's response to... 64 Cu-DOTA-TDP-2 uptake was significantly reduced (e.g. Figure 4 (As shown in B). Quantitative results showed that the SUV value and tumor / muscle ratio of the tumor in the blocking group were significantly lower than those in the non-blocking group at multiple examination time points, with the peak of the difference occurring 6 hours after probe injection, consistent with typical image results; conversely, important organs such as the liver, lungs, kidneys, and muscles showed significantly lower SUV values ​​and tumor / muscle ratios at multiple examination time points compared to the non-blocking group .... 64 Cu-DOTA-TDP-2 does not have the ability to specifically target and be taken up (e.g. Figure 4 (As shown in C). This illustrates that... 64 Cu-DOTA-TDP-2 has the specificity to target and bind to esophageal squamous cell carcinoma tumor tissue.

[0083] three, 64 In vitro imaging and biodistribution of Cu-DOTA-TDP-2:

[0084] In this invention, at the peak of probe uptake on PET imaging (i.e., 6 hours after probe injection), esophageal squamous cell carcinoma-bearing mice in both the non-blocking group (n=3) and the blocking group (n=3) were euthanized. Blood, heart, liver, spleen, kidney, lung, brain, intestine, pancreas, long bones, muscle, and tumors were collected from the tumor-bearing mice for ex vivo organ imaging. The results showed that the radioactive uptake of the tumors in the non-blocking group was significantly higher than that in the blocking group (e.g., ...). Figure 5 (As shown in A). Biodistribution results showed no significant difference in radioactive uptake in normal organs such as blood, heart, liver, spleen, kidney, lung, brain, intestine, pancreas, long bones, muscles, stomach, and skin between the non-blocking and blocking groups of tumor-bearing mice. Conversely, the percentage of radioactive uptake per gram of tumor tissue (%ID / g) in the non-blocking group was significantly higher than that in the blocking group (as shown in A). Figure 5 (as shown in B).

[0085] Example 3

[0086] This invention relates to novel radionuclide-targeted drugs. 177 Lu-DOTA-TDP-2, where 177 Lu is an isotope of lutetium with a half-life of 6.7 days. 177 The experimental principle of Lu-DOTA-TDP-2 targeting DDX24 helicase (e.g.) Figure 6 (as shown), i.e., radioactive nuclides 177 Lu synthesized a stable radionuclide-targeted drug by chelating the metal chelator DOTA with the peptide TDP-2. 177Lu-DOTA-TDP-2, this drug utilizes the specificity of TDP-2 to target and bind to the nucleolar DDX24 helicase, thereby releasing the radionuclide 177 Lu is directed into the cell nucleus, aided by... 177 The beta rays released during the decay of Lu directly kill tumor cells.

[0087] 177 Tagging and synthesis of Lu-DOTA-TDP-2:

[0088] Add 1.5 mg of the precursor DOTA-TDP-2 to 200 μl of sodium acetate buffer, then add 0.8 mL of [the solution is missing here]. 177 LuCl3 (12mCi, purchased from Guangzhou Atomic High-Tech Co., Ltd.) solution was used. The pH was adjusted to 4.5, and the reaction temperature was set at 37℃. After standing for 1 hour, the product was obtained, with the following structural formula: Figure 7 As shown.

[0089] Example 4

[0090] I. Cellular level characterization 177 Lu-DOTA-TDP-2 binding DDX24 activity

[0091] In order to characterize 177 To investigate the ability of Lu-DOTA-TDP-2 to target and bind to DDX24 esophageal squamous cell carcinoma cells, we performed competitive inhibition and saturation binding assays. In the competitive inhibition assay, EC109 cells were seeded in complete culture medium (5 × 10⁶ cells / well) in 96-well plates. 3 Cells / well were incubated overnight, and then the culture medium was converted to serum-free PRMI-1640. Different concentration gradients of DOTA-TDP-2 (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM) were added to each well for inhibition. Three replicates were set for each concentration gradient. The cells were then incubated at 37°C for 2 hours. After incubation, 5 μC was added to each well. 64 The Cu-DOTA-TDP-2 probe was incubated for another 2 hours. After incubation, the untaken material was washed three times with PBS. 64 Cu-DOTA-TDP-2 was used, and then the radioactivity in the cell pellet was counted using a gamma counter. Finally, GraphPad Prism was used to perform nonlinear regression fitting on the data. The results showed that DOTA-TDP-2 could block radioactivity in a dose-dependent manner. 177 The combination of Lu-DOTA-TDP-2 and DDX24, the IC of DOTA-TDP-2 50 The value was 68.6 ± 2.6 μM (e.g. Figure 8As shown in Figure A). In the cell saturation binding assay (48-well plate, 1×10⁻⁶ cells / well), 4 (cells / well), EC109 cell total binding experimental group added to each well 64 Cu-DOTA-TDP-2 (concentration gradients of 400 μM, 80 μM, 16 μM, 3.2 μM, 0.64 μM, and 0.13 μM); For the EC109 cell non-specific binding experimental group, add Cu-DOTA-TDP-2 to each well. 64 After Cu-DOTA-TDP-2 (concentration gradients of 400 μM, 80 μM, 16 μM, 3.2 μM, 0.64 μM, and 0.13 μM), 100-fold excess of unlabeled DOTA-TDP-2 was added to each well, followed by serum-free medium to a total volume of 100 μl per well. The mixture was incubated at 37°C for 2 h. After incubation, 100 μl of pre-cooled 0.01 M PBS (3 min × 2 times) was added to each well for washing. The washes were collected in plastic tubes, and the radioactivity count (CPM) per tube was measured using an automated gamma counter (PerkinElmer). The dissociation constant Kd was calculated using GraphPad Prism and Scatchard plots. The results showed... 177 Lu-DOTA-TDP-2's K d The value was 22.6 ± 5.9 μM (e.g. Figure 8 (As shown in B). The above results indicate that DOTA-TDP-2 is an effective ligand for binding DDX24 helicase.

[0092] two, 177 Lu-DOTA-TDP-2 in vitro stability test

[0093] This invention is based on radionuclides 177 Lu and peptide TDP-2 were used for radionuclide-targeted drug labeling synthesis, and radio-TLC was used for detection before labeling. 177 The radioactive peak of LuCl3 is visible, and the distance from the spotting point to the appearance of the radioactive peak is 100 mm (e.g., ...). Figure 9 (As shown in A); after labeling, radio-TLC was used to detect the products. 177 Lu-DOTA-TDP-2 was used to detect a radioactive peak at the baseline sampling point, with no other extraneous peaks observed (such as...). Figure 9 As shown in B), the prompt has been... 177 Lu was completely chelated to TDP-2; a portion of the product was left at room temperature for 144 hours and then subjected to radio-TLC again, and the radioactive peak was still visible at the baseline spot (e.g. Figure 9 (as shown in C), illustrating the labeled product. 177 Lu-DOTA-TDP-2 exhibits good stability.

[0094] III. Comparison of Cell Uptake and Blockade Experiments 177Differences in uptake of different peptides by esophageal squamous cell carcinoma cells after Lu labeling

[0095] To prove TDP-2 chelation 177 Lu has the highest specificity for targeting and binding to DDX24 helicase. This invention designs an equal-length scrambled peptide SDP (amino acid sequence: N-terminus - WVCARAQYAYTCCTY - carboxyl terminus) by scrambling the amino acid sequence of TDP-2. It also incorporates three peptides that specifically recognize DDX24 helicase that were previously screened and synthesized by the applicant's team (patent application filed, CN 110256551 A): a peptide derived from the p53 transcriptional active region (p53-1, amino acid sequence: N-terminus - SQETFSDLWKLLPEN - carboxyl terminus), a phosphorylated p53-based peptide (p53-2, amino acid sequence: N-terminus - S(p)QET(p)FSDLWKLLPEN - carboxyl terminus), and a peptide screened for Mdm2 / Mdmx-p53 in phage display experiments (Mdm2 / Mdmx-p53, amino acid sequence: N-terminus - LTFEHYWAQLTS - carboxyl terminus). Then, based on the above five targeting peptides, a polypeptide linked to the Arg-penetrating peptide RRRRRRRR was designed and synthesized, with the following amino acid sequences:

[0096] TDP-2: TVCTWYCYCAAYQRA-RRRRRRRR;

[0097] SDP: WVCARAQYAYTCCTY-RRRRRRRR;

[0098] p53-1: SQETFSDLWKLLPEN-RRRRRRRR;

[0099] p53-2: S(p)QET(p)FSDLWKLLPEN-RRRRRRRR;

[0100] Mdm2 / Mdmx-p53:LTFEHYWAQLTS-RRRRRRRR.

[0101] Based on the above five polypeptides that bind to penetrating peptides, further [they were] combined with [other peptides]. 177 Lu performed radionuclide labeling and synthesized five radionuclide-targeted drugs: 177 Lu-DOTA-TDP-2 177 Lu-DOTA-SDP, 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177Lu-DOTA-Mdm2 / Mdmx-p53 was prepared according to Example 3, and cell uptake experiments were performed on esophageal squamous cell carcinoma cells with high DDX24 expression. The experimental steps were as described in Step 1 of Example 2. It was found that... 177 Lu-DOTA-SDP, 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 177 Lu-DOTA-Mdm2 / Mdmx-p53 and 177 Compared to Lu, 177 The intake of Lu-DOTA-TDP-2 was highest at different time points (12h, 24h), and the differences were statistically significant (e.g., Figure 10 (As shown in A-B), this proves that at the cellular level... 177 Lu-DOTA-TDP-2 has the strongest ability to specifically target and bind to esophageal squamous cell carcinoma cells.

[0102] In addition, we conducted a cell blocking experiment, in which 100 μM of unlabeled DOTA-TDP-2 radiopeptide was added to each well of esophageal squamous cell carcinoma cells 2 hours in advance to conduct a competitive binding reaction, and then added separately after 2 hours. 177 Lu、 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 The experimental procedure and measurements for Lu-DOTA-Mdm2 / Mdmx-p53 were the same as those for cellular uptake experiments. Results showed that after TDP-2 blockade, esophageal squamous cell carcinoma cells showed [response to TDP-2]. 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 The uptake rate of Lu-DOTA-Mdm2 / Mdmx-p53 decreased by more than 50% compared to before non-blockade (e.g. Figure 11 As shown in A-B), the esophageal squamous cell carcinoma cells before and after the blockade... 177 Lu and 177 The uptake rate of Lu-DOTA-SDP did not change significantly, which indicates that 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177Lu-DOTA-Mdm2 / Mdmx-p53 exhibits the ability to specifically target and bind to esophageal squamous cell carcinoma cells at the cellular level. Furthermore, we found that after DOTA-TDP-2 blockade, esophageal squamous cell carcinoma cells showed increased binding to esophageal squamous cell carcinoma cells at different time points (12h and 24h). 177 Lu、 177 Lu-DOTA-TDP-2 177 Lu-DOTA-SDP, 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 There was no significant difference in the uptake rate of Lu-DOTA-Mdm2 / Mdmx-p53 (e.g. Figure 11 (As shown in A to B).

[0103] IV. Comparison of Cell Proliferation Inhibition Experiments 177 The inhibitory effect of Lu-labeled different peptides on the proliferation of esophageal squamous cell carcinoma cells

[0104] For comparison 177 To investigate the effect of different labeled peptides on the proliferation of esophageal squamous cell carcinoma cells after DOTA-TDP-2 blockade, we used a CCK-8 assay for cell proliferation inhibition. The experimental method was the same as the cell blockade assay: 100 μM of unlabeled DOTA-TDP-2 peptide was added to each well of the esophageal squamous cell carcinoma cells 2 hours prior to the competitive binding reaction. Two hours later, 5 μM of DOTA-TDP-2 peptide was added to each well in different blockade groups. 177 Lu、 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 Lu-DOTA-Mdm2 / Mdmx-p53 was used, with separate control groups (DOTA-TDP-2 only and no DOTA-TDP-2) set up. Cell proliferation inhibition was then assessed at 12 and 24 hours after incubation. Results showed that, compared with... 177 Lu, DOTA-TDP-2 177 Lu-DOTA-SDP, 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 Compared to Lu-DOTA-Mdm2 / Mdmx-p53 177 Lu-DOTA-TDP-2 significantly inhibited the proliferation of esophageal squamous cell carcinoma cells at different time points (e.g. Figure 12(As shown in A-B). The combined results of cell blocking and cell proliferation inhibition experiments show that: compared with the blank control group, the tumor cell proliferation ability of the blocking group with only DOTA-TDP-2 was not significantly reduced, indicating that DOTA-TDP-2 alone has no killing effect on tumor cells; however, after DOTA-TDP-2 blockade, esophageal squamous cell carcinoma cells showed no significant decrease in tumor cell proliferation. 177 Lu、 177 Lu-DOTA-SDP, 177 Lu-DOTA-TDP-2 177 Lu-DOTA-p53-1 177 Lu-DOTA-p53-2 and 177 With Lu-DOTA-Mdm2 / Mdmx-p53 uptake rates being essentially the same, 177 Lu-DOTA-TDP-2 significantly inhibited the proliferation of esophageal squamous cell carcinoma cells compared to the other five groups (inhibition rate exceeding the other five groups by more than 20 percentage points). Figures 11-12 As shown in the figure, this demonstrates that binding TDP-2 enhances the radionuclide. 177 Lu has the ability to inhibit tumor cell proliferation, while other peptides such as SDP with similar structures and targeting capabilities cannot achieve this effect.

[0105] Example 5

[0106] one, 177 Lu-DOTA-TDP-2 inhibits tumor growth in tumor-bearing mice.

[0107] This invention constructs a mouse model of esophageal squamous cell carcinoma and administers it via tail vein injection. 177 The efficacy of Lu-DOTA-TDP-2 in treating subcutaneous esophageal squamous cell carcinoma was evaluated. The experimental procedures were as follows: Figure 13 As shown in Figure A, 5-6 week old healthy Balb / c nude mice were randomly divided into four groups of nine mice each. Six days before the start of treatment (Day 6), an esophageal squamous cell carcinoma xenograft model was established by subcutaneous injection of EC109 cells into the right hind leg root of the mice. The treatment was divided into four groups: PBS group and low (5 MBq), medium (10 MBq), and high (15 MBq) doses of... 177 Lu-DOTA-TDP-2 was administered via tail vein injection. Treatment was repeated once, 6 days after the initial treatment, with an observation period of 36 days. At the end of the observation period (Day 36), a group of surviving tumor-bearing mice were photographed. Typical images (e.g.) Figure 13 As shown in Figure B, the tumor volume in tumor-bearing mice decreased with increasing treatment dose; the quantification results of tumor volume were consistent with those in typical images, and the tumor volume in the high-dose group was the smallest compared to the low- and medium-dose groups (e.g., ...). Figure 13 (As shown in C). Tumor growth curve results (e.g.) Figure 13As shown in Figure D, tumor growth was fastest in the PBS group, while that of the tail vein injection group was the fastest. 177 Lu-DOTA-TDP-2 significantly inhibited tumor growth, with the high-dose group showing the slowest tumor growth, followed by the medium-dose group. The low-dose group showed relatively rapid tumor growth, but still significantly slower than the PBS group. Survival curves of tumor-bearing mice (e.g.) Figure 13 As shown in Figure E, at the end of the experiment, the survival rates of tumor-bearing mice in the high, medium, and low dose groups were 88% (7 / 8), 63% (5 / 8), and 38% (3 / 8), respectively; while the median survival time of tumor-bearing mice in the PBS group was only 14 days, and all mice reached the study endpoint on day 19. These results demonstrate that tail vein injection of 15 MBq... 177 Lu-DOTA-TDP-2 can significantly inhibit tumor growth and prolong the survival of mice bearing esophageal squamous cell carcinoma.

[0108] II. High dose 177 Lu-DOTA-TDP-2 inhibits tumor proliferation in tumor-bearing mice and accelerates tumor cell apoptosis.

[0109] This invention aims to reveal high doses 177 The potential reasons for Lu-DOTA-TDP-2's inhibition of tumor growth were investigated through pathological examination. Immunohistochemical results showed that Ki67, a marker of tumor proliferation, was highly expressed in the PBS group, while in the high-dose group... 177 Lu-DOTA-TDP-2 expression was relatively low in tumor tissues after treatment (e.g. Figure 14 As shown in A), the Ki67 immunofluorescence staining results are consistent with the immunohistochemical staining results (as shown in A). Figure 14 (As shown in B). We also performed Caspase 3 staining to reflect differences in cell apoptosis among different dose groups. Immunohistochemical results showed that, compared with the PBS group, the high-dose group had significantly increased Caspase 3 expression (e.g., as shown in B). Figure 15 As shown in Figure A), immunofluorescence staining results also showed that the expression level of Caspase 3 in the high-dose group was significantly higher than that in the PBS group (as shown in Figure A). Figure 15 (As shown in B). The above results suggest that high doses... 177 Lu-DOTA-TDP-2 may achieve anti-tumor therapeutic effects by inhibiting the proliferation of esophageal squamous cell carcinoma tumors and accelerating tumor cell apoptosis.

[0110] III. In vivo assessment of tail vein injection 177 Security of Lu-DOTA-TDP-2

[0111] In order to clarify 177To assess the in vivo toxicity of Lu-DOTA-TDP-2, 5-6 week old healthy Balb / c nude mice were randomly divided into four groups of nine mice each. The grouping type and treatment methods were consistent with the efficacy assessment. On days 6, 12, and 36 after the first injection, three mice from each group were randomly selected. Fresh blood samples were rapidly collected after euthanasia to detect liver and kidney function. The indicators tested included alanine aminotransferase (ALT) and aspartate aminotransferase (AST), reflecting liver function, and blood urea nitrogen (BUN) and creatinine (Crea), reflecting kidney function. According to Charles River's experimental animal genetic quality control standards, the liver and kidney function indicators of all four groups of nude mice were within the normal physiological range (e.g., ...). Figure 16 (As shown in the image), and no abnormal deaths occurred. These results demonstrate that Balb / c nude mice can tolerate two consecutive tail vein injections of 15 MBq (D0, 6). 177 Treatment with Lu-DOTA-TDP-2 has a good safety profile.

[0112] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase, characterized in that: It is obtained by binding a radioactive nuclide to a polypeptide that targets the nucleolar DDX24 helicase; The polypeptide is TDP-2, and its amino acid sequence is TVCTWYCYCAAYQRARRRRRRRR; The radioactive nuclide mentioned is 177 Lu、 67 At least one of Cu; The drug mentioned is used to treat esophageal squamous cell carcinoma.

2. The method for preparing the novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase as described in claim 1, characterized in that... Includes the following steps: (1) Preparation of a polypeptide precursor for DOTA-targeting nucleolar DDX24 helicase; (2) Add the DOTA-targeting nucleolar DDX24 helicase polypeptide precursor to the buffer solution, then add the radionuclide, adjust the pH, and react to obtain a novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase. The amount of radionuclide added is 10-20 mCi of radionuclide per mg of DOTA-targeting nucleolar DDX24 helicase polypeptide precursor. The buffer solution is sodium acetate buffer solution; The pH adjustment mentioned above refers to adjusting the pH to 4-6; The reaction temperature is 35–50°C; The reaction time is 10–80 min.

3. The use of the novel radionuclide therapeutic drug targeting nucleolar DDX24 helicase as described in claim 1 in the preparation of drugs for treating esophageal squamous cell carcinoma or imaging products for esophageal squamous cell carcinoma.

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