A D-configuration oncolytic peptide-camptothecin conjugate and its preparation method and application

Abstract

This invention provides a class of D-configuration oncolytic peptide-camptothecin conjugates, their preparation methods, and applications, belonging to the fields of peptide preparation and biomedical technology. The steps are as follows: D-configuration peptides are synthesized using a solid-phase peptide synthesis method based on 9-fluorenemethoxycarbonyl. Then, the synthesized D-configuration peptides are covalently linked to a small molecule antitumor drug via a linker group. The D-configuration oncolytic peptide-camptothecin conjugates are obtained through peptide cleavage, separation and purification, and freeze-drying. The D-configuration oncolytic peptide-camptothecin conjugates significantly improve the solubility of camptothecin, exhibiting advantages such as high enzymatic stability, long half-life, and stronger inhibitory effect on tumor cell proliferation. Furthermore, this invention enables spatiotemporal monitoring of the release of camptothecin conjugates, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide preparation and biomedicine technology, specifically relating to a D-configuration oncolytic peptide-camptothecin conjugate, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer is one of the major threats to human health worldwide. Current traditional treatments for cancer include surgery, radiotherapy, and chemotherapy. Traditional anticancer drugs such as paclitaxel (PTX), camptothecin (CPT), and doxorubicin (DOX) have shown significant tumor-suppressive effects, but their limitations, such as low water solubility, strong systemic toxicity, and multidrug resistance, necessitate the development of novel and effective anticancer drugs with fewer side effects.

[0004] Camptothecin (CPT) is a small-molecule anticancer drug isolated from the bark and branches of the Camptotheca acuminata (also known as the Chinese camptotheca tree or the tree of happiness), native to China. CPT exhibits strong cytotoxicity and significant antitumor activity against various tumor cell lines. Studies have shown that DNA topoisomerase I (TopoI) is the target of CPT. Camptothecin compounds block the final step of the TopoI-DNA reaction—the recombination of single-stranded or double-stranded DNA at the cleavage site—leading to DNA breakage and cell death. However, CPT's high cytotoxicity to normal cells, poor water solubility, and weak cell membrane penetration limit its application.

[0005] Oncolytic peptides are bioactive peptides with tumor-suppressive effects. Compared with other drugs, they have many advantages in the treatment of tumors, such as small molecular weight, simple structure, high activity, high selectivity, fewer side effects, multiple administration methods, and low likelihood of inducing drug resistance. The antitumor activity of a peptide is related to its chain length, overall charge / hydrophobicity, helical structure, amino acid composition, and sequence. Oncolytic peptides are generally composed of a dozen to several dozen amino acid residues, are positively charged, and amphiphilic. By binding to the negatively charged tumor cell membrane, they can inhibit tumor cell proliferation at different stages of tumor cell growth and differentiation, achieving a specific killing effect on tumor cells and ultimately leading to tumor cell death.

[0006] LTX-315 (amino acid sequence: H-KKWWKKW-Dip-K-NH2), derived from the sequence of the antimicrobial peptide bovine lactoferrin, is a cationic oncolytic peptide that exhibits significant antitumor activity against tumor cell lines. LTX-315 disrupts mitochondrial and cell membranes, leading to the leakage of cellular contents, and can activate the body's own immune system to attack tumor cells. Through this dual mechanism, LTX-315 achieves highly efficient killing of tumor cells.

[0007] Aureins are a family of peptides composed of 13-25 amino acid residues, many of which exhibit anti-Gram-positive bacterial and anti-tumor activities. They were initially isolated from the skin secretions of the Australian bell frog. Experiments have shown that the 13-amino acid peptide Aurein 1.2 (amino acid sequence H-GLFDIIKKIAESF-NH2) exhibits significant anti-tumor activity against various conventional and drug-resistant tumor cell lines, with low toxicity to normal cells. Aurein 1.2 is a cationic amphiphilic peptide that induces neutral or anionic membrane lysis via a carpet mechanism, disrupting tumor cell membranes and mitochondrial membranes, causing cellular contents to leak out, triggering a strong immune response, and leading to cell death.

[0008] Traditional oncolytic peptides are L-configuration polypeptides, composed of L-amino acids and glycine. They can be extracted from nature, chemically synthesized, or recombinantly expressed. However, they suffer from poor stability, are easily degraded by proteases, have short half-lives, and possess potential immunogenicity. D-configuration polypeptides, composed of D-amino acids and glycine, do not exist in nature and can only be obtained through total chemical synthesis. Because the current central dogma cannot yield D-configuration polypeptides or D-configuration proteins, they are less easily recognized by the body's enzyme systems, exhibiting advantages such as low immunogenicity, good stability, and resistance to protease degradation.

[0009] The inventors discovered that Aurein 1.2 (H-GLFDIIKKIAESF-NH2) and LTX-315 (H-KKWWKKW-Dip-K-NH2), composed of L-amino acids and glycine, possess advantages such as high antitumor activity, broad anticancer spectrum, and low likelihood of inducing tumor drug resistance. However, Aurein 1.2 and LTX-315 are easily degraded by proteases, have short half-lives, and exhibit potential immunogenicity. This limits the widespread application of Aurein 1.2 and LTX-315. Summary of the Invention

[0010] Aurein 1.2 and LTX-315 suffer from poor stability, short half-life, and immunogenicity issues, while camptothecin exhibits high cytotoxicity to normal cells, poor water solubility, and weak cell membrane penetration. To address these shortcomings, this invention provides a D-configuration oncolytic peptide-camptothecin conjugate, its preparation method, and its applications. To address the drawbacks of L-type oncolytic peptides (easily degraded and immunogenic) and camptothecin (poor water solubility and weak cell membrane penetration), this invention employs a series of structural modifications to obtain a D-type oncolytic peptide composed of D-type amino acid residues. Further, a D-type oncolytic peptide-camptothecin conjugate covalently bound to the D-type oncolytic peptide and camptothecin is synthesized. The D-type oncolytic peptide is less susceptible to amino acid degradation, exhibits higher stability, and has lower immunogenicity. To address the high cost of D-isoleucine (D-Ile), this invention uses D-leucine (D-Leu) to replace D-Ile, significantly reducing the preparation cost of the oncolytic peptide. The D-type oncolytic peptide-camptothecin conjugate prepared in this experiment links camptothecin with a degradable ester bond, thereby increasing the inhibitory activity of the oncolytic peptide against tumor cells. Furthermore, this invention introduces a fluorescence-quenching group, dinitrophenyl (Dnp), into the D-type oncolytic peptide-camptothecin conjugate, utilizing the principle of intramolecular fluorescence self-quenching to monitor the release of camptothecin at both temporal and spatial levels. The dose-response curve for antitumor activity shows that the modified D-type oncolytic peptide-camptothecin conjugate exhibits increased solubility and significantly enhanced antitumor activity. Simultaneously, the time-response curve for antitumor activity indicates that the D-type oncolytic peptide-camptothecin conjugate greatly increases the stability of the oncolytic peptide and prolongs its action time. Therefore, the D-type oncolytic peptide-camptothecin conjugate of this invention has good practical application value.

[0011] Specifically, the present invention is achieved through the following technical solution:

[0012] In a first aspect, the present invention provides a D-type oncolytic peptide-camptothecin conjugate, said D-type oncolytic peptide-camptothecin conjugate comprising the following amino acid residue sequence:

[0013] YJF-599H-Glfkllkklaksf-NH2

[0014] YJF-600 CPT-AEEA-Glfkllkklaksf-NH2

[0015] YJF-601(CPT)2-k-AEEA-Glfkllkklaksf-NH2

[0016] YJF-602(CPT)2-kk(CPT)-AEEA-Glfkllkklaksf-NH2

[0017] YJF-603(CPT)2-kk(Dnp)-AEEA-Glfkllkklaksf-NH2

[0018] YJF-604CPT-AEEA-kkwwkkw-Dip-kk(Dnp)-NH2

[0019] YJF-605(CPT)2-k-AEEA-kkwwkkw-NH2

[0020] The aforementioned D-type oncolytic peptide-camptothecin conjugate significantly enhances the antitumor activity and stability of the oncolytic peptide. While disrupting the cell membrane, it is not degraded by proteases, allowing the conjugate to further penetrate the cell and release camptothecin. By simultaneously disrupting both the cell membrane and mitochondrial membrane, and blocking the rebinding of single-stranded or double-stranded DNA at the cleavage site, it induces DNA breakage, thus synergistically exerting its antitumor effect. Furthermore, the intramolecular fluorescence self-quenching effect of the combined use of Dnp and camptothecin enables spatiotemporal monitoring of camptothecin release.

[0021] In a second aspect, the present invention provides a method for preparing the above-mentioned D-type oncolytic peptide-camptothecin conjugate, the method comprising synthesizing a polypeptide using a solid-phase polypeptide synthesis method, and connecting the polypeptide with a linking group and a small molecule drug group by a condensation reaction to obtain the polypeptide.

[0022] Specifically, the above-mentioned oncolytic peptides were synthesized using a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenemethyloxycarbonyl.

[0023] A third aspect of the present invention provides the use of the above-described oncolytic peptide-camptothecin conjugate in the preparation of anticancer or antitumor drugs.

[0024] Preferably, the cancer or tumor may be selected from breast cancer, nasopharyngeal carcinoma, thymic carcinoma, bladder cancer, bronchogenic carcinoma, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, ganglioneuroma, gastric cancer, ganglioneuroma, papillary thyroid carcinoma, squamous cell carcinoma of the head and neck, and testicular cancer.

[0025] A fourth aspect of the present invention provides the use of the above-mentioned D-type oncolytic peptide-camptothecin conjugate drug release monitoring agent. The oncolytic peptide-camptothecin conjugate of the present invention, used in conjunction with camptothecin, can emit specific blue fluorescence after camptothecin release, thereby achieving spatiotemporal monitoring of camptothecin release. It can be applied to cellular, tissue, and in vivo animal imaging, and further explore the antitumor activity and mechanism of action of the D-type oncolytic peptide-camptothecin conjugate at the molecular, cellular, tissue, and animal levels.

[0026] The beneficial technical effects of the above technical solution are as follows:

[0027] 1. The above technical solution can significantly improve the antitumor activity of oncolytic peptides. The D-type oncolytic peptide-camptothecin conjugate synthesized in this invention enhances the inhibitory activity of oncolytic peptides against tumor cells. Specifically, the IC50 of each peptide... 50 (24h) results are as follows: In Jurkat cells, 14.4±2.7 μM (YJF-599), 10.9±2.3 μM (YJF-600), 17.9±1.2 μM (YJF-601), 10.4±2.1 μM (YJF-605); in A20 cells, 19.3±2.2 μM (YJF-599), 4.5±1.8 μM (YJF-600), 7.9±2.4 μM (YJF-601), 18.3±3.9 μM (YJF-602), 7.9±3.3 μM (YJF-605); in PC-3 cells, 44.5± The concentrations of oncolytic peptides were 5.5 μM (YJF-599), 21.7 ± 1.2 μM (YJF-600), 26.1 ± 1.1 μM (YJF-601), 38.6 ± 5.5 μM (YJF-602), and 48.9 ± 1.1 μM (YJF-605) in Hela-S3 cells, and finally 76.1 ± 5.6 μM (YJF-599), 11.8 ± 2.8 μM (YJF-600), 14.7 ± 3.2 μM (YJF-601), and 10.8 ± 2.2 μM (YJF-605), which were significantly improved compared with the original oncolytic peptides LTX-315 and Aurein 1.2.

[0028] 2. The above technical solution can significantly improve the stability of oncolytic peptides. Previously developed oncolytic peptides such as Aurein1.2 and LTX-315 are composed of L-amino acids, which are easily degraded by proteases in vivo, resulting in insufficient stability, short half-lives, and high immunogenicity, making them easily rejected by the body. This project uses more stable D-amino acids to synthesize novel D-type oncolytic peptides. D-type oncolytic peptides have high enzymatic stability, are more stable than traditional L-type oncolytic peptides, can significantly prolong the antitumor effect time and improve antitumor activity, while reducing their immunogenicity, and have significant application value.

[0029] 3. The above technical solution can significantly reduce the synthesis cost of oncolytic peptides. The previously developed Aurein1.2 peptide sequence contains three isoleucine residues. D-isoleucine (D-Ile) is expensive, resulting in high synthesis costs for D-configuration oncolytic peptides. This project replaces D-Ile with D-leucine (D-Leu), significantly reducing the synthesis cost of the peptide without affecting its antitumor activity.

[0030] 4. The above technical solution achieves spatiotemporal monitoring of the release and distribution of small molecule antitumor drugs through intramolecular self-quenching. By combining Dnp with camptothecin, camptothecin emits blue fluorescence upon release, enabling temporal and spatial monitoring of camptothecin release. This can be applied to cellular, tissue, and in vivo animal imaging, further exploring the antitumor activity and mechanism of action of oncolytic peptide-camptothecin conjugates at the molecular, cellular, tissue, and animal levels.

[0031] 5. The above technical solution can significantly reduce the toxicity of camptothecin to normal cells and enhance its solubility. By covalently linking camptothecin to the polypeptide, the water solubility of camptothecin is increased by more than 6 times (calculated by molar amount).

[0032] In summary, the previously developed oncolytic peptides Aurein 1.2 and LTX-315 have high synthesis costs, poor enzymatic stability, short half-lives, high immunogenicity, and relatively low antitumor activity. To avoid these drawbacks, this invention designs and synthesizes a novel D-type oncolytic peptide-camptothecin conjugate, which has advantages such as high enzymatic stability, long half-life, low immunogenicity, and stronger activity. Furthermore, by combining Dnp with camptothecin, the release of camptothecin results in the emission of blue fluorescence, enabling temporal and spatial monitoring of camptothecin release, thus showing promising application prospects. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 A comparison diagram of the characteristics of L-type and D-type polypeptides;

[0035] Figure 2 This is a schematic diagram of the solid-phase polypeptide synthesis method of the present invention;

[0036] Figure 3 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-599 of this invention are shown below.

[0037] Figure 4 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of YJF-600 of this invention are shown below.

[0038] Figure 5 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-601 of this invention are shown below.

[0039] Figure 6The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-602 of this invention are shown below.

[0040] Figure 7 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-603 of this invention are shown below.

[0041] Figure 8 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of YJF-604 of this invention are shown below.

[0042] Figure 9 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of YJF-605 of this invention are shown below.

[0043] Figure 10 A schematic diagram illustrating the mechanism of intramolecular self-quenching monitoring of camptothecin release;

[0044] Figure 11 shows the cellular-level evaluation of the D-type oncolytic peptide-camptothecin conjugate and its combination therapy according to the present invention. 50 ;

[0045] Figure 12 This invention aims to evaluate the inhibitory effect of the D-type oncolytic peptide-camptothecin conjugate on tumor cell proliferation at the cellular level.

[0046] Figure 13 This invention aims to evaluate at the cellular level the inhibitory effect of the combined use of D-type oncolytic peptide-camptothecin conjugate on tumor cell proliferation.

[0047] Figure 14 This is the time-dependent curve of the D-type oncolytic peptide-camptothecin conjugate of the present invention inhibiting tumor cell proliferation.

[0048] Figure 15 This study aims to monitor the stability of the L-type and D-type oncolytic peptides of the present invention in serum.

[0049] Figure 16 This invention utilizes the intramolecular self-quenching principle to monitor the release of camptothecin. Detailed Implementation

[0050] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. In the specific embodiments, all original reagents and raw materials are commercially available. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] As mentioned earlier, Aurein 1.2 and LTX-315 are composed of L-type amino acids, which have disadvantages such as low enzymatic stability, short half-life, high immunogenicity, and high cost, thus limiting their widespread application.

[0052] In view of this, in a typical embodiment of the present invention, a D-type oncolytic peptide-camptothecin drug conjugate is provided, wherein the oncolytic peptide-peptide drug conjugate comprises the following amino acid residue sequence:

[0053] YJF-599H-Glfkllkklaksf-NH2

[0054] YJF-600 CPT-AEEA-Glfkllkklaksf-NH2

[0055] YJF-601(CPT)2-k-AEEA-Glfkllkklaksf-NH2

[0056] YJF-602(CPT)2-kk(CPT)-AEEA-Glfkllkklaksf-NH2

[0057] YJF-603(CPT)2-kk(Dnp)-AEEA-Glfkllkklaksf-NH2

[0058] YJF-604CPT-AEEA-kkwwkkw-Dip-kk(Dnp)-NH2

[0059] YJF-605(CPT)2-k-AEEA-kkwwkkw-NH2

[0060] The above-mentioned oncolytic peptide-drug conjugate can significantly improve the enzymatic stability of oncolytic peptides, reduce synthesis costs, and enhance their antitumor activity. At the same time, by using Dnp in combination with camptothecin, camptothecin emits blue fluorescence after release, enabling temporal and spatial monitoring of camptothecin release.

[0061] In another specific embodiment of the present invention, a method for synthesizing the above-mentioned D-type oncolytic peptide-camptothecin conjugate is provided, the method comprising: synthesizing a polypeptide using a solid-phase polypeptide synthesis method; and linking the polypeptide with a linking group and a small molecule drug group through a condensation reaction.

[0062] Specifically, the above-mentioned polypeptides were synthesized using a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenemethyloxycarbonyl.

[0063] Unless otherwise specified, this invention uses Rink Amide Am resin (with a degree of substitution of 0.38 mmol / g) to synthesize polypeptides containing amide terminals; the amino acids used in the synthesis process, except for glycine (Gly, G), are all Fmoc-D-type amino acids.

[0064] More specifically, the method for synthesizing the D-type oncolytic peptide-camptothecin conjugate includes:

[0065] Step 1: Synthesize D-type polypeptides using a solid-phase polypeptide synthesis method based on 9-fluorenemethyloxycarbonyl.

[0066] Step 2: The synthesized polypeptide is attached with a linker group, a small molecule drug group, through a condensation reaction;

[0067] Step 3: Add peptide cleavage reagent to the condensation product to cleave the condensation product off the resin;

[0068] Step 4: The peptide-cleaved product was separated, purified, and freeze-dried to obtain the D-type oncolytic peptide-camptothecin conjugate.

[0069] The specific experimental method for step 1 is as follows:

[0070] Weigh Rink-Amide AM resin, pre-activate and activate the resin, then remove the Fmoc protecting group, and perform amino acid condensation according to the amino acid sequence. After all amino acid condensation is completed, wash the resin and wash it again after removing the last protecting group.

[0071] In another specific embodiment of the present invention, the above-mentioned pre-activation and activation experimental methods are as follows: rinsing with DMF and DCM alternately, and soaking in DMF at room temperature for 1-2 hours. Soaking the resin in a DMF / DCM mixed solution (1:1, volume ratio, v:v) and reacting at 28°C in a constant temperature shaker for 0.5 hours.

[0072] The experimental method for removing the Fmoc protecting group was as follows: at 28°C, the protecting group was removed using a DMF solution (v:v) containing 20% ​​piperidine. The removal was performed twice, for 5 minutes and 10 minutes respectively.

[0073] The condensation reaction experiment method was as follows: each amino acid was condensed twice at 28℃ for 30 minutes and 40 minutes respectively. The ratio of reactants was Fmoc-D-type amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents.

[0074] The cleaning method was as follows: rinsed alternately with DMF and DCM, and then the residual solvent was removed by pumping water.

[0075] The specific experimental method for step 2 is as follows:

[0076] The D-type oncolytic peptide synthesized in step 1 is condensed with the AEEA linker group, and then condensed with a small molecule drug group. The reactant ratios for YJF-600 are camptothecin:HATU:HOAT:DIEA = 2 equivalents: 1.8 equivalents: 2 equivalents: 4 equivalents (molar ratio); for YJF-601, camptothecin:HATU:HOAT:DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio); for YJF-602, camptothecin:HATU:HOAT:DIEA = 4 equivalents: 3.6 equivalents: 4 equivalents: 8 equivalents (molar ratio); and for YJF-603, camptothecin:HATU:HOAT:DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio).

[0077] The specific experimental method for step 3 is as follows:

[0078] Add peptide-cleaving reagent to the prepared condensation product in a ratio of TFA:phenol:water:TIPS = 88:5:5:2 (v:v:v:v). React in a shaker at 28°C in the dark for 1.5–2 hours. Wash the resin twice with 0.5 mL of TFA. Concentrate the reaction product to 3 mL using high-purity nitrogen. Add pre-cooled anhydrous diethyl ether to the concentrate to precipitate the target peptide. Centrifuge to obtain the crude peptide, and repeat centrifugation three times with anhydrous diethyl ether. Dry the product in a fume hood.

[0079] The specific experimental method for step 4 is as follows:

[0080] The crude peptide product obtained in step 3 was dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and then analyzed and identified by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and ESI-MS. The dissolved crude peptide was stored at -80°C for later use. It was then freeze-dried to obtain flocculent crude peptide. The crude peptide was further dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and then purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected pure peptide solution was stored at -80°C overnight, and then freeze-dried to obtain the target pure peptide.

[0081] In another specific embodiment of the present invention, the application of the above-mentioned oncolytic peptide-camptothecin conjugate in the preparation of anticancer or antitumor drugs is provided.

[0082] In another specific embodiment of the present invention, the present invention can treat solid tumors and hematologic malignancies. Solid tumors may include breast cancer, nasopharyngeal carcinoma, thymic carcinoma, bladder cancer, bronchogenic carcinoma, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, ganglioneuroma, gastric cancer, ganglioneuroma, papillary thyroid carcinoma, squamous cell carcinoma of the head and neck, testicular cancer, and liver cancer. Hematologic malignancies may include acute myeloid leukemia, acute lymphoblastic leukemia, granulocytic leukemia, and malignant lymphoma.

[0083] In another specific embodiment of the present invention, the above-mentioned D-type oncolytic peptide-camptothecin conjugate is provided as a drug release monitoring agent. The oncolytic peptide-drug conjugate of the present invention, using Dnp in combination with camptothecin, can emit blue fluorescence after camptothecin release, thereby achieving spatiotemporal monitoring of camptothecin release. It can be applied to cellular, tissue, and in vivo animal imaging, and further explore the antitumor activity and mechanism of action of the oncolytic peptide-drug conjugate at the molecular, cellular, tissue, and animal levels.

[0084] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0085] Example 1

[0086] In this embodiment, all target peptides were prepared using solid-phase peptide synthesis technology based on 9-fluorene methoxycarbonyl (Fmoc-SPPS). Unless otherwise specified, Rink Amide Am resin (degree of substitution 0.38 mmol / g) was selected to synthesize peptides containing amide terminals; the amino acids used in the synthesis process, except for glycine (Gly, G), which is a non-chiral amino acid, were all Fmoc-D-type amino acids.

[0087] The scale of peptide synthesis is typically 0.15 mmol. The basic process of peptide synthesis is as follows: Figure 1 As shown.

[0088] Solid-phase peptide synthesis experiment: 400 mg of Rink Amide AM resin (1 equivalent) was weighed and rinsed alternately with DMF and DCM. The resin was pre-activated by soaking in DMF at room temperature for 1–2 h. After another alternating rinse, the resin was soaked in a DMF / DCM mixed solution (1:1, v:v) and shaken at 28 °C in a constant temperature shaker for 0.5 h to activate the resin. Then, the Fmoc protecting group was removed using DMF (v:v) containing 20% ​​piperidine, with two removal cycles of 5 minutes and 10 minutes respectively. Each amino acid underwent two condensation cycles at 28 °C for 30 minutes and 40 minutes respectively. The reactant ratio was Fmoc-D-type amino acid: HCTU: DIPEA = 3 equivalents: 2.8 equivalents: 6 equivalents. After the condensation of the last amino acid, its Fmoc protecting group was removed, and the resin was rinsed alternately with DMF and DCM.

[0089] Synthesis of D-type oncolytic peptide-camptothecin conjugate: The synthesized oncolytic peptide and the linking group are condensed through an amide bond, and then the linking group is condensed with a small molecule drug group through an amide bond. The reactant ratios for YJF-600 are: camptothecin: HATU: HOAT: DIEA = 2 equivalents: 1.8 equivalents: 2 equivalents: 4 equivalents (molar ratio); for YJF-601, camptothecin: HATU: HOAT: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio); for YJF-602, camptothecin: HATU: HOAT: DIEA = 4 equivalents: 3.6 equivalents: 4 equivalents: 8 equivalents (molar ratio); and for YJF-603, camptothecin: HATU: HOAT: DIEA = 3 equivalents: 2.8 equivalents: 3 equivalents: 6 equivalents (molar ratio).

[0090] Peptide cleavage and purification: Peptide cleavage reagent was added to the prepared condensation product in a ratio of TFA:phenol:water:TIPS = 88:5:5:2 (v:v:v:v). The reaction was carried out in a shaker at 28°C for 1.5–2 h. The resin was washed twice with 0.5 mL of TFA, and the reaction product was concentrated to 3 mL with high-purity nitrogen. Pre-cooled anhydrous diethyl ether was added to the concentrate to precipitate the target peptide. The crude peptide was obtained by centrifugation, and the process was repeated three times with the addition of anhydrous diethyl ether. The product was dried in a fume hood. The crude peptide product obtained in step 3 was then dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA. The crude peptide was then analyzed and identified by analytical reversed-phase high-performance liquid chromatography and ESI-MS. The dissolved crude peptide was stored at -80°C for later use. It was then freeze-dried to obtain flocculent crude peptide. The crude peptide was dissolved in a mixed solution of acetonitrile and water containing 1‰ TFA, and then separated and purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected pure peptide solution was placed at -80°C overnight and then freeze-dried to obtain the target pure peptide.

[0091] The chemical structural formulas, primary amino acid sequences, analytical reversed-phase high-performance liquid chromatography (RP-HPLC) chromatograms, and ESI-MS mass spectra of the seven D-type oncolytic peptide-camptothecin conjugates prepared in this invention are shown below. Figure 3-9 As shown.

[0092] CCK-8 assay for tumor cell proliferation inhibition:

[0093] A20, Jurkat, Hela S3, and PC-3 cells in logarithmic growth phase were selected, collected, and counted before being seeded in 96-well plates. After seeding, the plates were sterilized, and the cells were incubated for 24 hours. Then, 50 μL of YJF-599, YJF-600, YJF-601, YJF-602, YJF-605, CPT, LTX-315, and Aurein 1.2 were added to each well at final concentrations of (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM), with three replicates per group. The cells were cultured for another 24 hours. After 24 hours, CCK-8 (final concentration 5 μmol·L⁻¹) was added in the dark. -1 Add the solution to each well, mix gently, and incubate in an incubator for 4 hours to terminate the culture. Measure the absorbance at a wavelength of 450 nm, repeat the measurement three times, record the results, and perform subsequent calculations according to the formula.

[0094] like Figure 12 As shown, the novel D-type oncolytic peptide-camptothecin conjugate can kill A20, Jurkat, Hela S3, and PC-3 tumor cells in a concentration-dependent manner. The IC50 of the novel D-type oncolytic peptide-camptothecin conjugate is... 50The values ​​were significantly improved compared to Aurein 1.2 and LTX-315, and the antitumor activity was significantly enhanced.

[0095] D-type oncolytic peptide-camptothecin combination therapy inhibits tumor cell proliferation:

[0096] A20, Jurkat, Hela S3, and PC-3 cells in logarithmic growth phase were selected, collected, and counted before being seeded in 96-well plates. After seeding, the plates were sterilized, and the cells were incubated for 24 hours. Three drug groups were set up: Group 1 consisted of a 1:1 mixture of YJF-599 and CPT; Group 2 consisted of a fixed YJF-599 concentration of 12.5 μM with the addition of CPT solutions at concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM; and Group 3 consisted of a fixed CPT concentration of 12.5 μM with the addition of YJF-599 solutions at concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. Each group had three replicates, and the cells were cultured for another 24 hours. After 24 hours, CCK-8 (final concentration 5 μmol·L⁻¹) was added under light-protected conditions. -1 Add the solution to each well, mix gently, and incubate in an incubator for 4 hours to terminate the culture. Measure the absorbance at a wavelength of 450 nm, repeat the measurement three times, record the results, and perform subsequent calculations according to the formula.

[0097] like Figure 13 As shown, the covalently linked YJF-599 and CPT (i.e., YJF-600) have a stronger inhibitory effect on tumor cells than the combination of the two drugs after non-covalent linkage.

[0098] Time-dependent assay of the D-type oncolytic peptide-camptothecin conjugate's inhibitory effect on tumor cell proliferation:

[0099] A20 cells in logarithmic growth phase were selected, collected, and counted before being seeded in 96-well plates (100 μl per well). 50 μL of YJF-599, YJF-600, YJF-601, YJF-602, YJF-605, CPT, LTX-315, and Aurein 1.2 were added to a final concentration (10 μM), with three replicates per group. Cells were cultured for 4 h, 12 h, 24 h, 36 h, 48 h, and 72 h in an incubator. After the specified time, CCK-8 (final concentration 5 μmol·L⁻¹) was added under light. -1 Add the absorbance to each well, mix gently, and incubate in an incubator for 4 hours to terminate the culture. Measure the absorbance at a wavelength of 450 nm, repeating the measurement three times.

[0100] The results are as follows Figure 14As shown, the D-type oncolytic peptide-camptothecin conjugate at 10 μM exhibited a 65% inhibition rate against A20 cells at 12 h, while LTX-315 and Aurein 1.2 showed inhibition rates of approximately 25%-30%, indicating that the D-type peptide possesses higher antitumor activity. The D-type oncolytic peptide-drug conjugate reached its peak activity at 36-48 h. LTX-315 and Aurein 1.2 reached their peak activity at 12 h and 36 h (inhibition rates of 30% and 35%, respectively), respectively, after which their activity began to decline, reaching only 20% inhibition rate at 72 h. Both the D-type oncolytic peptide-camptothecin conjugate and the D-type oncolytic peptide-camptothecin conjugate maintained their antitumor activity for an extended period. These results demonstrate that the D-type oncolytic peptide-camptothecin conjugate possesses better antitumor activity and stability.

[0101] Serum stability test:

[0102] Prepare a 5% serum solution by dissolving serum in PBS. Weigh appropriate amounts of Aurein 1.2, LTX-315, YJF-600-1(H-AEEA-Glfkllkklaksf-NH2), and YJF-605-1(H-AEEA-kkwwkkw-NH2) peptide solids, dissolve them in water, and add the prepared serum solution to the peptide solution to prepare a mixed solution with a final concentration of 200 μM. Shake and stir three times. Immediately take 80 μl of the reaction solution, add 20 μl of acetonitrile containing 0.1% TFA, shake three times, and let stand on ice for 2 minutes. Then add 60 μl of acetonitrile and 400 μl of deionized water, each containing 0.1% TFA, shake three times, and immediately store at -80℃. The reaction tube was sealed and incubated in a constant temperature water bath at 37°C for 72 hours. Samples were taken at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, and 72 hours, respectively, following the above method.

[0103] The results are as follows Figure 15 As shown, the remaining percentages of Aurein 1.2 and LTX-315 peptides gradually decreased with increasing time, and all were hydrolyzed within 48 hours. In contrast, the YJF-600-1 and YJF-605-1 conjugates were less prone to hydrolysis, with remaining percentages of 80% and 70%, respectively, at 72 hours. These results indicate that D-type oncolytic peptides are more stable than L-type oncolytic peptides.

[0104] Experiments using the intramolecular self-quenching principle to monitor the release of camptothecin in real time:

[0105] Prepare a 5% serum solution by dissolving serum in PBS. Weigh appropriate amounts of YJF-604 and camptothecin solid, dissolve them in water, and add the prepared serum solution to the PBS solution to prepare a mixed solution with a final concentration of 200 μM. Shake and stir three times. Immediately take 80 μl of the reaction solution, add 20 μl of acetonitrile containing 0.1% TFA, shake three times, and let stand on ice for 2 minutes. Then add 60 μl of acetonitrile and 400 μl of deionized water, each containing 0.1% TFA, shake three times, and immediately store at -80℃. Seal the reaction tube and incubate it in a constant temperature water bath at 37℃ for 72 h. Take samples at 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 36 h, 48 h, and 72 h. Monitor the release of camptothecin in real time using RP-HPLC and fluorescence spectrophotometry.

[0106] The results are as follows Figure 16 As shown, with increasing time, RP-HPLC and spectrophotometry detection revealed a significant enhancement in the hydrolysis peak of YJF-604, while the fluorescence intensity of YJF-604 also increased, reaching almost the same fluorescence intensity as the camptothecin monomer at 72 h. These results indicate that introducing DNP into the D-configuration oncolytic peptide-camptothecin conjugate allows for spatiotemporal monitoring of camptothecin release.

[0107] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A D-type oncolytic peptide-camptothecin conjugate, characterized in that, The D-type oncolytic peptide-camptothecin conjugate is: YJF-600 CPT-AEEA-Glfkllkklaksf-NH2.

2. The method for preparing the D-type oncolytic peptide-camptothecin conjugate according to claim 1, characterized in that, The preparation method of the D-type oncolytic peptide-camptothecin conjugate is as follows: A peptide was synthesized using a solid-phase peptide synthesis method based on 9-fluorenemethyloxycarbonyl. The synthesized peptide was then linked to a linking group and a small molecule drug group through a condensation reaction. A peptide-cleaving reagent was added to the condensation product to cleave the condensation product from the resin. The cleaved product was identified, separated, and purified to obtain a D-type oncolytic peptide-camptothecin conjugate.

3. The use of the D-type oncolytic peptide-camptothecin conjugate according to claim 1 in the preparation of antitumor drugs; The tumors were selected from prostate cancer, acute lymphoblastic leukemia, malignant lymphoma, and cervical cancer.

4. A pharmaceutical composition, characterized in that, The composition comprises the D-type oncolytic peptide-camptothecin conjugate as described in claim 1.

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