Nectin-4-targeting bicyclic peptide nuclide ligand and probe, preparation method therefor and use thereof

Abstract

Provided are a Nectin-4-targeting bicyclic peptide nuclide ligand and probe, a preparation method therefor, and a use thereof. The bicyclic peptide nuclide ligand and the bicyclic peptide nuclide probe are used for the imaging diagnosis of a Nectin-4-positive tumor patient and can achieve medication guidance for a patient receiving treatment with an anti-cancer targeted drug and real-time efficacy monitoring. A pharmacokinetic modification molecular polysarcosine is added between a chelating agent for radionuclide labeling and a Nectin-4-targeting polypeptide, thereby improving the biocompatibility of the probe and optimizing pharmacokinetic properties, especially non-tumor tissue clearance kinetics, so as to achieve better diagnosis and treatment effects. In the bicyclic peptide nuclide ligand DOTA-Sar-bicyclic peptide, DOTA is used as a bifunctional chelating agent, thereby achieving better in vivo and in vitro stability.

Description

A bicyclic peptide ligand and probe targeting Nectin-4, its preparation method and application Technical Field

[0001] This invention belongs to the field of nuclear medicine technology, specifically relating to a bicyclic peptide nuclide ligand, probe, preparation method and application of Nectin-4. Background Technology

[0002] In my country, the number of cancer cases and deaths is rising year by year. According to the 2022 global cancer data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, my country had 4.82 million new cancer cases and 2.57 million cancer deaths in 2022, far exceeding other countries. Early and accurate detection of lesions throughout the body using nuclear medicine imaging technology can provide clinicians with better guidance for treatment. Currently, molecular imaging modalities mainly include: molecular magnetic resonance imaging, optical imaging, targeted ultrasound, single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Among these, nuclear medicine imaging (PET, SPECT) plays an increasingly important role in clinical practice due to its high sensitivity, strong tissue penetration, ability to perform in vivo quantification, and the availability of multiple radionuclides.

[0003] Nectin-4 (gene name PVRL4, poliovirus receptor 4) protein belongs to the Nectin family of the immunoglobulin superfamily. The Nectin family works in conjunction with cadherins to significantly influence the generation and maintenance of adhesive and tight junctions, regulating various cellular behaviors including cell adhesion, growth, differentiation, migration, and apoptosis. Nectin-4 protein is specifically expressed in the embryo and placenta, expressed in a few normal adult tissues (including skin), and upregulated in certain cancer patients, such as urothelial carcinoma, breast cancer, ovarian cancer, and pancreatic cancer; its overexpression is associated with tumor progression and poor clinical prognosis. Therefore, exploring targeted Nectin-4 protein and combination therapies may provide more treatment options for patients with advanced solid tumors.

[0004] Enfortumab vedotin is an FDA-approved targeted therapy for Nectin-4 positive (urothelial carcinoma) tumors, demonstrating efficacy in both early and advanced (metastatic) tumors. However, while some patients are sensitive to Enfortumab vedotin, others develop varying degrees of resistance after a period of targeted therapy. Due to the long treatment cycle of anti-Nectin-4 targeted therapy, the high cost of antibody drugs, and the tendency to develop resistance, designing novel molecular probes targeting the Nectin-4 site in breast cancer is crucial for screening Nectin-4 positive tumor patients, real-time efficacy monitoring during Enfortumab vedotin treatment, and designing personalized precision medicine. Literature reports… 68 Compared with the probe of this invention, Ga-N188 has limitations when applied to Nectin-4 positive tumor models. The research of this invention confirms that the bicyclic polypeptide (FDSH polypeptide) has excellent Nectin-4 targeting performance and can effectively distinguish the Nectin-4 expression status of different tumor cells. Summary of the Invention

[0005] This invention provides a bicyclic peptide nuclide ligand and probe targeting Nectin-4, as well as their preparation method and application. The ligand and probe can specifically target Nectin-4 positive tumors and be used for imaging diagnosis of Nectin-4 positive tumor patients, enabling medication guidance and real-time efficacy monitoring for patients receiving targeted anticancer drug therapy.

[0006] The technical solution adopted by the present invention to achieve the above objectives is: a bicyclic peptide nucleoside ligand targeting Nectin-4, having the structure shown in formula (I):

[0007] In formula (I): Sar is the pharmacokinetic modifier polysarcosine; L is a metal chelating agent.

[0008] Furthermore, L is selected from DOTA, Nota, HYNIC, TPA, DTPA, NODAGA, TETA, and PCTA.

[0009] Or DOTAM.

[0010] Furthermore, L stands for DOTA.

[0011] Furthermore, the degree of polymerization of the Sar is 1-10, preferably 10.

[0012] This invention discloses a method for preparing the above-mentioned bicyclic peptide nuclide ligand, comprising the following steps:

[0013] (1) Before the synthesis reaction, the α-amino group and side chain functional group of the amino acid in the bicyclic peptide are protected. The protected amino acid needs to be further activated. Then, the bicyclic peptide is condensed in dimethyl sulfoxide or N,N-dimethylformamide by solid-phase synthesis to synthesize DOTA-Sar-peptide-resin.

[0014] (2) In a mixture of TFA, EDT, TIS and water, the DOTA-Sar-peptide-resin obtained in step (1) is subjected to resin cleavage reaction, and the resulting DOTA-Sar-peptide is reacted in TATA solution to obtain DOTA-Sar-bicyclic peptide ligand.

[0015] This invention discloses a bicyclic peptide nuclide probe targeting Nectin-4, wherein the bicyclic peptide nuclide probe comprises a bicyclic peptide nuclide ligand and a radionuclide; the bicyclic peptide nuclide ligand is the aforementioned bicyclic peptide nuclide ligand or a bicyclic peptide nuclide ligand obtained by the aforementioned preparation method.

[0016] Furthermore, selected from 99m Tc, 68 Ga、 64 Cu、 111 In、 90 Y、 225 Ac、 137 Cs、 90 Sr、 125 I, 131 I, 153 Sm、 18 F, 177 Any one of Lu;

[0017] Furthermore, the radionuclide is 68 Ga;

[0018] This invention discloses a method for preparing the above-mentioned bicyclic peptide nuclide probe, comprising the following steps:

[0019] A mixture containing ammonium acetate and the chelating agent Sar-bicyclic peptide was prepared, a radioactive nuclide solution was added, the mixture was heated to react, and after the reaction was completed, it was cooled to room temperature to prepare a bicyclic peptide nuclide probe.

[0020] This invention also discloses the application of the above-mentioned bicyclic peptide isotope ligand, the bicyclic peptide isotope ligand obtained by the above-mentioned preparation method, the above-mentioned bicyclic peptide isotope probe, or the bicyclic peptide isotope probe obtained by the above-mentioned preparation method in evaluating the efficacy of targeted drug treatment for Nectin-4 related diseases, including urothelial carcinoma, breast cancer, pancreatic cancer, bladder cancer, lung cancer, ovarian cancer, and other tumors that can affect Nectin-4 expression changes.

[0021] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least:

[0022] First, the bicyclic peptide nuclide ligand and bicyclic peptide nuclide probe of the present invention can be used for imaging diagnosis of Nectin4-positive tumor patients, and can realize medication guidance and real-time efficacy monitoring for patients receiving anticancer drugs, especially Nectin-4 targeted therapy.

[0023] Secondly, the bicyclic peptide nuclide ligand DOTA-Sar-bicyclic peptide of the present invention incorporates the pharmacokinetic modification molecule polysarcosine between the chelating agent used for radionuclide labeling and the Nectin-4-targeted polypeptide, thereby improving the biocompatibility of the probe and optimizing its pharmacokinetic properties, especially the clearance kinetics of non-tumor tissues, to achieve better diagnostic and therapeutic effects.

[0024] Third, the DOTA-Sar-bicyclic peptide ligand of this invention uses DOTA as a bifunctional chelating agent, giving it better in vitro and in vivo stability. (Theoretical derivation) 68 The technical effects that Ga-NOTA can achieve 18 F-NOTA can achieve a similar effect. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0026] Figure 1 shows the MS and HPLC spectra of DOTA-Sar10-FDSH, where (A) is the MS spectrum and (B) is the HPLC spectrum.

[0027] Figure 2 is 68 Radio HPLC analysis chromatogram of Ga-DOTA-Sar10-FDSH;

[0028] Figure 3 is 68 In vitro stability analysis of Ga-DOTA-Sar10-FDSH;

[0029] Figure 4 shows the injection 68 PET / CT images of NSG mouse MDA-MB-468 breast cancer model after 0.5h, 1h, 2h and 3h of Ga-DOTA-Sar10-FDSH treatment;

[0030] Figure 5 shows the injection 68PET / CT images of NSG mouse MDA-MB-468, MCF-7, and MDA-MB-231 breast cancer models after 0.5 h of Ga-DOTA-Sar10-FDSH treatment;

[0031] Figure 6 shows the injection 68 Data graphs of %ID / g in NSG mouse MDA-MB-468 breast cancer model after 0.5h, 1h, 2h and 3h of Ga-DOTA-Sar10-FDSH;

[0032] Figure 7 shows the injection 68 %ID / g data of NSG mouse MCF-7 breast cancer model after 0.5h of Ga-DOTA-Sar10-FDSH;

[0033] Figure 8 shows the injection 68 Data graph of %ID / g in NSG mouse MDA-MB-213 breast cancer model after 0.5h of Ga-DOTA-Sar10-FDSH;

[0034] Figure 9 shows the injection 68 PET / CT images of NSG mouse HT-1376 bladder cancer model after 1h, 2h and 3h of Ga-DOTA-Sar10-FDSH treatment;

[0035] Figure 10 shows the injection 68 In vivo blood clearance analysis diagram in the MDA-MB-468 breast cancer model after Ga-DOTA-Sar10-FDSH;

[0036] Figure 11 is 68 Ga-N188 and 68 Comparative analysis of PET / CT imaging effects of Ga-DOTA-Sar10-FDSH. Detailed Implementation

[0037] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0038] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Example 1: Preparation of a bicyclic peptide ligand targeting Nectin-4

[0040] The bicyclic peptide (Cys-Pro-1Nal-D-Asp-Cys-Met-HomoArg-Asp-D-Trp-Ser-Thr-Pro-Hyp-Trp-Cys(TATA Cyclized)) monomer and DOTA-Sar10-FDSH precursor were synthesized by China Zhongtai Biochemical Co., Ltd.

[0041] The preparation method of the bicyclic peptide nuclide ligand includes the following steps:

[0042] (1) Before the synthesis reaction, the α-amino group and side chain functional groups of the amino acids in the bicyclic peptide are protected by 9-fluoromethyl carbonyl (Fmoc) to prevent side reactions between amino acids. The protected amino acids need to be activated in a mixture of carbon tetrachloride (DCC) and diisopropyl carbamate (DIC) (volume ratio 1:1). Then, the bicyclic peptide is condensed in dimethyl sulfoxide solution by solid phase synthesis. After deprotection, purification and identification, DOTA-Sar-peptide-resin is prepared.

[0043] (2) The DOTA-Sar-peptide-resin obtained in step (1) was subjected to resin cleavage reaction at room temperature for 5-10 min in a mixture of trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT), triisopropylsilane (TIS) and water (volume ratio of 90:5:2.5:2.5) to obtain the DOTA-Sar-peptide. The reaction was carried out via a three-stage Michael addition of cysteine ​​residues in the DOTA-Sar-peptide to 1,1',1”-(1,3,5-triazine-1,3,5-triyl)tripropyl-2-en-1-one (TATA) (mass-volume ratio of DOTA-Sar-peptide to TATA is 1 mg:1 mL) under alkaline conditions to obtain the cyclic product DOTA-Sar-bicyclic peptide ligand.

[0044] The MS and HPLC chromatograms of DOTA-Sar10-FDSH are shown in Figure 1.

[0045] Example 2: Preparation of a bicyclic peptide nuclide probe targeting Nectin-4

[0046] 68 Preparation of Ga-DOTA-Sar10-FDSH:

[0047] Take 44 μL of 1.5 M ammonium acetate solution and slowly add it to a solution containing 10 μL of the 1 μg / μL dicyclic peptide ligand DOTA-Sar10-FDSH. Then add 400 mL of freshly rinsed radionuclide solution. 68 The reaction was carried out using GaCl3 at 75°C under a heating mantle for 20 minutes. After the reaction was completed, the mixture was cooled to room temperature to prepare a bicyclic peptide nuclide probe targeting Nectin-4. 68 The specific reaction route for Ga-DOTA-Sar10-FDSH is shown below:

[0048] right 68 Radioactive HPLC analysis of Ga-DOTA-Sar10-FDSH was performed using an Agilent 1260 HPLC system equipped with a YMC-Pack ODS-A analytical column (250 × 4.6 mm L.DS-5 μm, 12 nm). The gradient elution time was 20 min, and the flow rate was 1 mL / min. Mobile phase A was deionized water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA). The elution gradient was initially set to 75% A and 25% B, and after 20 min, it was 25% A and 75% B. The results are shown in Figure 2. As can be seen from Figure 2, 68 The labeling efficiency of Ga-DOTA-Sar10-FDSH was >95%, and the radiochemical purity after purification by Sep-Pak C18 column was >98%.

[0049] Example 3: In vitro stability experiment of the bicyclic peptide nuclide probe targeting Nectin-4

[0050] Take a certain amount 68 Ga-DOTA-Sar10-FDSH was added to physiological saline, and its radiochemical purity and stability were measured at different time points. The results are shown in Figure 3. After 1 h, 2 h, and 3 h, the radiochemical purity and stability of Ga-DOTA-Sar10-FDSH were determined. 68 The radiochemical purity of Ga-DOTA-Sar10-FDSH remains greater than 98%, maintaining good stability.

[0051] Example 4: PET Imaging Study

[0052] SPF-grade NSG female mice, 5 weeks old, were provided by Shanghai Yaokang Biotechnology Co., Ltd. After two days of acclimatization in the animal facility, MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer cells were injected subcutaneously into the right axilla of the mice at a volume of 0.1 mL (5 × 10⁶ cells / mL dispersed in HBSS). The mice were then fed for 2-3 weeks post-injection until the solid tumor reached 500-800 mm in size. 3 It is used for imaging experiments. 100-150 μCi / 0.2 mL is injected via the tail vein. 68 Ga-DOTA-Sar10-FDSH was used in small animal PET imaging experiments at 0.5h, 1h, 2h, and 3h post-injection. The imaging results are shown in Figures 4 and 5. The results indicate that Nectin-4 positive MDA-MB-468 tumors... 68 The Ga-DOTA-Sar10-FDSH compound exhibits high uptake, with clearly visible tumor uptake and no gallbladder uptake. The peptide remains at the tumor site for a longer period, which is more beneficial for tumor diagnosis. In the MDA-MB-468 breast cancer tumor model, 68 Tumor uptake of Ga-DOTA-Sar10-FDSH was clearly visible, with no gallbladder uptake. The peptide remained at the tumor site for a longer time, which is more conducive to tumor diagnosis (Figure 4). In the MDA-MB-231 breast cancer tumor model group, tumor uptake... 68 The significant decrease in Ga-DOTA-Sar10-FDSH indicates that the probe of this invention specifically binds to the Nectin-4 site.

[0053] Further quantitative results in the MDA-MB-468 breast cancer tumor model showed that injection 68 0.5 hours after Ga-DOTA-Sar10-FDS H, tumor uptake 68 The ID% / g value of Ga-DOTA-Sar10-FDSH was 3.78 ± 0.34 (Figure 6); in the MCF-7 and MDA-MB-231 breast cancer tumor models, tumor uptake... 68 The ID% / g values ​​of Ga-DOTA-Sar10-FDSH were 3.35±0.45 and 1.15±0.25, respectively (Figures 7 and 8).

[0054] Example 5 68 PET Imaging Study of Ga-DOTA-Sar10-FDSH in Tumor-Bearing Mice (Bladder Cancer)

[0055] SPF-grade NSG female mice, 5 weeks old, were provided by Shanghai Yaokang Biotechnology Co., Ltd. After two days of acclimatization in the animal facility, HT-1376 human bladder cancer cells were injected subcutaneously into the right axilla of the mice. The injection volume was 0.1 mL (5 × 10⁻⁶). 6 (cells / mL dispersed in HBSS), and continued feeding for 2-3 weeks after injection until the solid tumor mass grows to 500-800 mm. 3 It is used for imaging experiments. 100-150 μCi / 0.2 mL is injected via the tail vein. 68 Ga-DOTA-Sar10-FDSH was used in small animal PET imaging experiments at 1, 2, and 3 hours post-injection. The imaging results are shown in Figure 9. The results indicate that Nectin-4 positive HT-1376 tumors... 68 The Ga-DOTA-Sar10-FDSH compound has a high uptake rate.

[0056] Example 5 68 Pharmacokinetic Analysis of Ga-DOTA-Sar10-FDSH in Tumor-Bearing Rats

[0057] NSG mice carrying MDA-MB-468 breast cancer tumors were randomly divided into several groups, with 3 mice in each group. Mice in each group were injected via tail vein. 68 Ga-DOTA-Sar10-FDSH was administered to animals at 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, and 180 min post-injection. Blood samples were collected, weighed, and radioactivity counts were measured. After decay correction, the percentage of injected dose per gram of blood (%ID / g) was calculated. The results showed that... 68 The half-life of Ga-DOTA-Sar10-FDSH in blood clearance is approximately 17 min (Figure 10).

[0058] Literature reports 68 In a Nectin-4 positive tumor model, Ga-N188 was injected... 68 One hour after Ga-DOTA-Sar10-FDSH administration, tumor uptake was below 0.8% ID / g, significantly lower than that of the compound in this embodiment. This result indicates that the compound in this embodiment exhibits superior PET / CT imaging for tumor visualization and imaging contrast compared to the compound in this embodiment. 68 Ga-N188 (Figure 11). Therefore, compared to 68 Ga-N188, this patented compound, is theoretically able to better visualize tumors in tumor diagnosis, potentially revealing smaller and more tumor lesions, thus bringing greater clinical diagnostic value.

[0059] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A bicyclic peptide ligand targeting Nectin-4, characterized in that, It has the structure shown in equation (I): In formula (I): Sar is the pharmacokinetic modifier polysarcosine; L is a metal chelating agent.

2. The bicyclic peptide nuclide ligand as described in claim 1, characterized in that, The L is selected from DOTA, Nota, HYNIC, TPA, DTPA, NODAGA, TETA, PCTA, or DOTAM.

3. The bicyclic peptide nuclide ligand as described in claim 2, characterized in that, L stands for DOTA.

4. The bicyclic peptide nuclide ligand as described in claim 1, characterized in that, The degree of polymerization of the Sar is 1-10, preferably 10.

5. The method for preparing the bicyclic peptide nuclide ligand according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Before the synthesis reaction, the α-amino group and side chain functional group of the amino acid in the bicyclic peptide are protected. The protected amino acid needs to be further activated. Then, the bicyclic peptide is condensed in dimethyl sulfoxide or N,N-dimethylformamide by solid-phase synthesis to synthesize DOTA-Sar-peptide-resin. (2) In a mixture of TFA, EDT, TIS and water, the DOTA-Sar-peptide-resin obtained in step (1) is subjected to resin cleavage reaction, and the resulting DOTA-Sar-peptide is reacted in TATA solution to obtain DOTA-Sar-bicyclic peptide ligand.

6. A bicyclic peptide nuclide probe targeting Nectin-4, characterized in that, The bicyclic peptide nuclide probe comprises a bicyclic peptide nuclide ligand and a radionuclide; the bicyclic peptide nuclide ligand is the bicyclic peptide nuclide ligand according to any one of claims 1-4 or the bicyclic peptide nuclide ligand obtained by the preparation method according to claim 5.

7. The bicyclic peptide nuclide probe as described in claim 6, characterized in that, The radionuclides are selected from 99m Tc, 68 Ga、 64 Cu、 111 In、 90 Y、 225 Ac、 137 Cs、 90 Sr、 125 I, 131 I, 153 Sm、 18 F, 177 Any one of Lu.

8. The bicyclic peptide nuclide probe as described in claim 7, characterized in that, The radioactive nuclide is 68 Ga.

9. The method for preparing the bicyclic peptide nuclide probe according to any one of claims 6-8, characterized in that, Includes the following steps: A mixture containing ammonium acetate and the chelating agent Sar-bicyclic peptide was prepared, a radioactive nuclide solution was added, the mixture was heated to react, and after the reaction was completed, it was cooled to room temperature to prepare the bicyclic peptide nuclide probe.

10. The application of the bicyclic peptide nuclide ligand as described in any one of claims 1-4, the bicyclic peptide nuclide ligand obtained by the preparation method of claim 5, the bicyclic peptide nuclide probe as described in any one of claims 6-8, or the bicyclic peptide nuclide probe obtained by the preparation method of claim 9 in evaluating the efficacy of targeted drug therapy for Nectin-4 related diseases, wherein the Nectin-4 related diseases include urothelial carcinoma, breast cancer, pancreatic cancer, bladder cancer, lung cancer, and ovarian cancer, as well as other tumors that can affect changes in Nectin-4 expression.

Images