A novel cyclic polypeptide, its preparation method and application in pancreatic cancer diagnostic drugs

Abstract

The application discloses a novel cyclic polypeptide and a preparation method and application thereof in a pancreatic cancer diagnosis drug, and belongs to the technical field of fluorescence contrast agents. The application provides a cyclic polypeptide, which has an amino acid sequence Cyclo(KVWRVLFNSPD) and is annular at the head and tail through an alpha-amino group of an N-terminal lysine and an alpha-carboxyl group of a C-terminal aspartic acid. The cyclic polypeptide can be covalently coupled with a near-infrared fluorescent dye to form a pancreatic cancer targeting near-infrared fluorescent molecular probe with a structure general formula as shown in M-L-G, wherein M is a light labeling group, L is a linking group, and G is a targeting ligand TG-6. The application further provides a preparation method of the cyclic polypeptide and the fluorescent probe. Animal experiment results show that the probe can specifically target pancreatic cancer tissues, has high tumor uptake and strong intratumoral retention capacity, has high tumor / normal tissue fluorescence contrast, and can be applied to real-time visual navigation in pancreatic cancer surgery, assisting doctors in accurately defining tumor boundaries and improving surgical accuracy.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent contrast agent technology, specifically relating to a novel cyclic polypeptide, its preparation method, and its application in pancreatic cancer diagnostic drugs. Background Technology

[0002] Malignant tumors have long ranked among the top causes of disease burden globally, posing a significant public health challenge that seriously threatens human life and health. Statistics show that approximately 27,000 people die from cancer every day worldwide, meaning that nearly ten million lives are lost to cancer each year. Faced with this grim situation, accurate early screening and subsequent personalized, effective treatment measures have been proven to be crucial in reducing cancer mortality and improving prognosis.

[0003] In recent years, although clinical treatment methods for cancer have become increasingly diverse, with significant advancements in chemotherapy, radiotherapy, targeted therapy, and immunotherapy, surgical resection remains the core treatment strategy for most solid tumors. It is particularly noteworthy that for some localized or early-stage solid tumors, radical resection may even be the only way to achieve a cure. The fundamental principle of oncological surgery lies in balancing two main goals: on the one hand, striving for complete removal of the primary lesion and metastatic lesions to minimize the risk of postoperative recurrence; on the other hand, preserving as much surrounding normal tissue structure as possible, such as nerves, blood vessels, lymphatic vessels, and functional organs, to avoid unnecessary functional damage or complications caused by excessive resection.

[0004] However, accurately distinguishing tumor tissue from healthy tissue has long been a technical challenge in actual surgical procedures. This is especially true in patients with early-stage or advanced-stage cancer, where numerous tiny lesions lacking typical morphological characteristics (such as micrometastases and nodules) are often present. These tiny lesions differ very little from the surrounding normal tissue in color, texture, and boundaries, making accurate identification and boundary delineation difficult for surgeons to achieve solely through visual observation and tactile judgment. This limitation can easily lead to two adverse consequences: insufficient resection results in residual lesions and recurrence; excessive resection damages important healthy tissue, impacting the patient's postoperative quality of life.

[0005] To overcome these challenges, surgical navigation systems have emerged and continue to evolve. Currently, surgical navigation systems based on optical molecular imaging principles can achieve real-time, dynamic imaging monitoring of tumors and surrounding lesions. These systems possess both structural and functional imaging capabilities, continuously acquiring tissue image data from multiple angles under high-throughput conditions, providing surgeons with visualized and quantified lesion boundary information. With advancements in optical imaging devices and image processing algorithms, the importance of optical surgical navigation systems in guiding precise tumor resection during surgery has become increasingly prominent, making them one of the crucial auxiliary tools in modern precision surgery.

[0006] However, despite the increasing maturity of system hardware and imaging technology, the development of supporting optical molecular probes has lagged behind. In particular, optical molecular probes suitable for pancreatic cancer, possessing high targeting specificity, high sensitivity, and good biocompatibility, are still scarce. Therefore, developing novel optical molecular probes with pancreatic cancer targeting capabilities has become one of the key bottlenecks in promoting the clinical translation and application expansion of optical surgical navigation technology. Summary of the Invention

[0007] In view of this, the present invention aims to provide a novel cyclic polypeptide, its preparation method, and its application in pancreatic cancer diagnostic drugs. Through systematic screening, the present invention has discovered that the novel cyclic polypeptide TG-6 possesses the potential for precise in vivo imaging of pancreatic cancer. Subsequent small animal in vivo imaging experiments have confirmed that near-infrared fluorescent molecular probes constructed based on TG-6 can specifically target and identify various pancreatic cancer tissues, with extremely low uptake in normal tissues, demonstrating great promise for assisting surgeons in accurately defining tumor boundaries during surgery.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a cyclic polypeptide TG-6 with the amino acid sequence Cyclo(KVWRVLFNSPD), wherein the cyclic polypeptide is cyclically formed end-to-end by the α-amino group of the N-terminal lysine and the α-carboxyl group of the C-terminal aspartic acid.

[0009] This invention also provides a method for preparing the above-mentioned cyclic polypeptide TG-6, comprising the following steps: A solid-phase synthesis strategy using Fmoc was employed, with Wang resin as a carrier. Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus to assemble the linear peptide H2N-Lys-Val-Trp-Arg-Val-Leu-Phe-Asn-Ser-Pro-Asp-OAll. After removing the N-terminal Fmoc protecting group and the C-terminal OAll protecting group, head-to-tail cyclization was performed in solution. Finally, the cyclic peptide was obtained through cleavage and purification.

[0010] The present invention also provides a fluorescent molecular probe, characterized in that it comprises the above-mentioned cyclic polypeptide TG-6.

[0011] Preferably, its structure is shown in the general formula MLG; wherein, M is a photolabeling group; G is a targeting ligand, the targeting ligand being a cyclic polypeptide TG-6; and L is a linking group.

[0012] Preferably, the photolabel is selected from organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules.

[0013] Furthermore, the optical marker is selected from near-infrared I fluorescent dyes MPA, IRDye800, 800cw, Cy7.5, ICG, Cy5.5 or near-infrared II fluorescent dyes; more preferably, the fluorescent dye is selected from MPA, IRDye800, Cy7.5.

[0014] Preferably, L is selected from any one of 6-aminohexanoic acid, PEG3, PEG4, PEG6, or G6; each with the following structural formula:

[0015] As a preferred option, any of the following structures may be selected: .

[0016] This invention also provides a method for preparing the near-infrared fluorescent molecular probe MPA-TG-6, comprising the following steps: The near-infrared fluorescent dye MPA-COOH was fully activated by weighing 1.0 molar amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS). After complete activation of MPA-COOH, an equimolar amount of pure TG-6 peptide was dissolved and reacted with MPA-NHS, and 5 molar amounts of N,N-diisopropylethylamine (DIPEA) were added. After the reaction was completed, the MPA-TG-6 reaction solution was purified by semi-preparative liquid chromatography, and the liquid with qualified purity was collected, lyophilized, and confirmed by ESI-MS as the target product.

[0017] This invention also provides the application of the above-mentioned cyclic polypeptide TG-6 or fluorescent molecular probe in the preparation of diagnostic drugs or reagents for pancreatic cancer.

[0018] Preferably, the applications include: precise localization of pancreatic cancer tissue boundaries or fluorescence imaging in intraoperative navigation for pancreatic cancer, and molecular imaging diagnostic imaging for pancreatic cancer.

[0019] It contains at least the following beneficial technical effects: 1. Systematic screening revealed that the novel cyclic peptide TG-6 possesses the potential for precise in vivo imaging of pancreatic cancer. Subsequent small animal in vivo imaging experiments confirmed that near-infrared fluorescent molecular probes constructed based on TG-6 can specifically target and identify various pancreatic cancer tissues, with extremely low uptake in normal tissues, demonstrating significant application potential in assisting surgeons to accurately define tumor boundaries during surgery. Furthermore, as a peptide drug, TG-6 has outstanding advantages such as simple synthesis, easy structural modification, strong cell penetration, and low immunogenicity, which contrasts sharply with the shortcomings of existing pancreatic cancer antibody-drug conjugates, including complex synthesis processes, long in vivo metabolic times, poor tissue penetration, and potential immunogenic risks.

[0020] 2. In vivo optical imaging results have confirmed that the near-infrared fluorescent molecular probe constructed based on TG-6 exhibits excellent targeting effects against various pancreatic cancers. This novel fluorescent probe's ability to specifically target tumor sites makes it a promising candidate for precise intraoperative navigation in pancreatic cancer surgery.

[0021] This invention uses the near-infrared fluorescent dye MPA, which has better in vivo stability and hydrophilicity, as an optical imaging group, thereby improving the pharmacokinetics of the drug in vivo. Attached Figure Description

[0022] Figure 1 The structural formula of the novel cyclic polypeptide TG-6 is shown.

[0023] Figure 2 This is a mass spectrometry analysis result of the novel cyclic polypeptide TG-6.

[0024] Figure 3 The structure of the near-infrared fluorescent probe MPA-TG-6 is shown.

[0025] Figure 4 This is a mass spectrometry analysis result of the near-infrared fluorescent probe MPA-TG-6.

[0026] Figure 5 This study aims to provide in vivo and in vitro fluorescence imaging and quantitative fluorescence analysis of the near-infrared fluorescent probe MPA-TG-6 in pancreatic cancer BxPc-3 and CAPAN-1 tumor-bearing mice. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0033] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0034] Example 1 Solid-phase synthesis of a novel cyclic polypeptide TG-6 Weigh 250 mg of Wang resin with a loading of 0.1 mmol / g and place it in a 5 mL dichloromethane reaction tube, allowing it to swell at room temperature for 20 min. Then, using DMF as the solvent, prepare a 5 mL solution of Fmoc-Asp-OAll, HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate), and DIPEA (also 5 times the molar amount of the resin), pre-activating for 10 min. Add this activated solution to the reaction tube and couple it with the resin at room temperature for 60 min. After the reaction is complete, remove the reaction solution and wash the resin four times with DMF.

[0035] To block unreacted active sites, 5 mL of acetic anhydride solution containing DIPEA (10 times the molar amount of resin) was added to the reaction tube, and the mixture was blocked at room temperature for 30 min. This blocking procedure was repeated once. After blocking, the resin was deprotected with Fmoc by 5 mL of 50% morphine DMF solution for 10 min, and this process was repeated twice. After each deprotection, the resin was washed with DMF four times.

[0036] Subsequently, following the standard solid-phase synthesis cycle, subsequent amino acids were coupled sequentially. Taking Fmoc-Pro-OH as an example: Fmoc-Pro-OH, HCTU, and DIPEA (each 5 times the molar amount of resin) were dissolved in DMF to prepare a 5 mL solution. After pre-activation for 10 min, the resin was added, and the reaction was carried out at room temperature for 60 min. After the reaction, the mixture was washed four times with DMF. The reaction progress was monitored at each coupling and deprotection step using the ninhydrin assay. A negative result was considered a complete reaction. This cycle was repeated sequentially from the C-terminus to the N-terminus until the full-length assembly of the sequence H2N-Lys-Val-Trp-Arg-Val-Leu-Phe-Asn-Ser-Pro-Asp-OAll was completed.

[0037] After coupling was completed, the reaction solution was dried, and the resin was washed four times with DMF. OAll deprotection was then performed: 15 mg of tetrakis(triphenylphosphine)palladium was weighed, 100 μL of phenylsilane was added, and the volume was adjusted to 5 mL with dichloromethane. This solution was added to the resin, and the reaction was allowed to proceed for 2 h. This deprotection process was repeated twice. Afterward, the resin was repeatedly washed with a saturated sodium diethyldithiocarbamate solution in DMF until the washings were colorless to remove palladium residue. The N-terminal Fmoc protecting group was then removed again with a morpholine / DMF solution. PyBOP and DIPEA (each 5 times the molar amount of the resin) were dissolved in DMF to prepare a 5 mL solution, which was added to the resin and allowed to react overnight at room temperature to achieve cyclization. After cyclization, the reaction solution was dried, washed six times with DMF and three times with methanol, and then thoroughly dried.

[0038] Finally, 5 mL of lysis buffer (trifluoroacetic acid: triisopropylsilane: water = 95:2.5:2.5, volume ratio) was added to the linear-cyclic peptide resin, and the mixture was stirred at room temperature for 3 hours. The filtrate was collected by filtration, concentrated under reduced pressure, and then pre-cooled to -20°C methyl tert-butyl ether was added at a volume ratio of 1:4 (concentrate to methyl tert-butyl ether). The mixture was centrifuged to precipitate, and the supernatant was discarded. The resulting solid was the crude peptide after removing all side-chain protecting groups. The crude peptide was purified by semi-preparative high-performance liquid chromatography and characterized by ESI-MS to finally obtain the target peptide TG-6, the structural formula of which is shown below. Figure 1 The mass spectrometry analysis results are shown below. Figure 2 ESI-MS results: [M+H] += 1342.95, [M+2H] 2+ = 672.22, which is consistent with the theoretical molecular weight.

[0039] First, 0.5 mg of the near-infrared dye MPA-COOH was weighed out, and condensing agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added in a ratio of 1.5 molar amounts relative to the dye. Both were dissolved in 50 μL of DMF, and the mixture was activated at 35 °C in the dark for 1 hour to convert the dye carboxyl groups into an active ester intermediate. Then, an equimolar amount of the pure cyclic peptide TG-6 was weighed out and dissolved in 50 μL of DMF. This peptide solution was added to the above activation reaction solution, along with 5 molar amounts relative to MPA-COOH. N,N Using diisopropylethylamine (DIPEA) as a basic catalyst, the reaction was continued at 35 °C in the dark for 0.5 hours, causing coupling between the active ester and the amino group of the polypeptide to form an MPA-cyclic peptide conjugate. After the reaction, the resulting reaction solution was directly purified by semi-preparative high-performance liquid chromatography (HPLC). The eluent fractions of acceptable purity were collected according to the target peak, combined, and lyophilized to obtain the target product. Finally, ESI-MS confirmed that the molecular ion peak of the product was [M-3H]. 3- = 749.90, consistent with the theoretical value, confirming that the obtained compound is MPA-TG-6, structural formula see Figure 3 The mass spectrometry analysis results are shown in Figure 4 .

[0040] Example 2 Fluorescence imaging and quantitative fluorescence analysis of the prepared near-infrared fluorescent probe MPA-TG-6 in in vivo and in vitro of pancreatic cancer BxPc-3 tumor-bearing mice. Before probe injection, tumor-bearing model mice underwent their first imaging (recorded as 0 h), and this 0 h imaging result was used as the control baseline for subsequent experiments. The prepared near-infrared fluorescent probe MPA-TG-6 was prepared as a saline solution, and 0.15 mL (approximately 15 nM) was injected into the tail vein of three nude mice bearing pancreatic cancer BxPc-3 tumors. Near-infrared fluorescence imaging was performed using an IVIS SpectrumLumina K (Series III) in vivo imaging system, with excitation and emission wavelengths set to 780 nm and 845 nm, respectively. Optical signals were acquired at different time points after drug administration. Through this continuous imaging monitoring, the distribution of the fluorescent drug in the model mice and its enrichment in the tumor region were observed. After completing in vivo imaging monitoring, the tumor-bearing model mice were euthanized and immediately dissected, and tumor tissue and major organs such as the heart, liver, spleen, lungs, kidneys, stomach, pancreas, small intestine, and colon / rectum were separated. The excised tissues were arranged sequentially for in vitro fluorescence imaging to observe the distribution of the probe in each organ. Semi-quantitative analysis of the fluorescence signals in each tissue and organ was performed using Living Image v 4.2 software. In vitro and in vivo fluorescence imaging results in tumor-bearing mice showed that the probe MPA-TG-6 could rapidly and accurately target pancreatic cancer (BxPc-3) tissue with high tumor / normal tissue contrast.

[0041] Example 3 Fluorescence imaging and quantitative analysis of the prepared near-infrared fluorescent probe MPA-TG-6 in in vivo and in vitro in CAPAN-1 pancreatic cancer-bearing mice. Before probe injection, tumor-bearing model mice underwent their first imaging (recorded as 0 h), and this 0 h imaging result was used as the control baseline for subsequent experiments. The prepared near-infrared fluorescent probe MPA-TG-6 was prepared as a saline solution, and 0.15 mL (approximately 15 nM) was injected into the tail vein of three nude mice bearing pancreatic cancer CAPAN-1 tumors. Near-infrared fluorescence imaging was performed using an IVIS SpectrumLumina K (Series III) in vivo imaging system, with excitation and emission wavelengths set to 780 nm and 845 nm, respectively. Optical signals were acquired at different time points after drug administration. Through this continuous imaging monitoring, the distribution of the fluorescent drug in the model mice and its enrichment in the tumor region were observed. After completing in vivo imaging monitoring, the tumor-bearing model mice were euthanized and immediately dissected, and tumor tissue and major organs such as the heart, liver, spleen, lungs, kidneys, stomach, pancreas, small intestine, and colon / rectum were separated. The excised tissues were arranged sequentially for in vitro fluorescence imaging to observe the distribution of probes within each organ. Semi-quantitative analysis of the fluorescence signals in each tissue and organ was performed using Living Image v 4.2 software. See [link to documentation]. Figure 5In vitro and in vivo fluorescence imaging results in tumor-bearing mice showed that the probe MPA-TG-6 could rapidly and accurately target pancreatic cancer (CAPAN-1) tissue with high tumor / normal tissue contrast.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cyclic polypeptide TG-6, characterized in that, Its amino acid sequence is Cyclo(KVWRVLFNSPD), wherein the cyclic polypeptide is cyclically formed end-to-end by the α-amino group of its N-terminal lysine and the α-carboxyl group of its C-terminal aspartic acid.

2. The method for preparing the cyclic polypeptide TG-6 according to claim 1, characterized in that, Includes the following steps: A solid-phase synthesis strategy using Fmoc was employed, with Wang resin as a carrier. Fmoc-protected amino acids were sequentially coupled from the C-terminus to the N-terminus to assemble the linear peptide H2N-Lys-Val-Trp-Arg-Val-Leu-Phe-Asn-Ser-Pro-Asp-OAll. After removing the N-terminal Fmoc protecting group and the C-terminal OAll protecting group, head-to-tail cyclization was performed in solution. Finally, the cyclic peptide was obtained through cleavage and purification.

3. A fluorescent molecular probe, characterized in that, Includes the cyclic polypeptide TG-6 as described in claim 1.

4. The fluorescent molecular probe according to claim 3, characterized in that, Its structure is shown in the general formula MLG; where M is the photolabeling group; G is the targeting ligand, which is a cyclic polypeptide TG-6; and L is the linker group.

5. The fluorescent molecular probe according to claim 4, characterized in that, The optical label is selected from organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules.

6. The fluorescent molecular probe according to claim 4, characterized in that, The L is selected from any one of 6-aminohexanoic acid, PEG3, PEG4, PEG6 or G6.

7. The fluorescent molecular probe according to claim 3, characterized in that, Choose from any of the following structures: 。 8. The use of the cyclic polypeptide TG-6 of claim 1 or the fluorescent molecular probe of claim 3 in the preparation of diagnostic drugs or reagents for pancreatic cancer.

9. The application according to claim 7, characterized in that, The applications include: precise localization of pancreatic cancer tissue boundaries or fluorescence imaging in intraoperative navigation for pancreatic cancer, and molecular imaging diagnostic imaging for pancreatic cancer.

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