Polypeptide conjugate and application thereof in preparation of medicine for treating colorectal cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide conjugate. According to the polypeptide conjugate disclosed by the invention, a series of polypeptide conjugate (PDCs) compounds are synthesized by taking ligand polypeptide of GC-C as a carrier and carrying cytotoxin through precise modification; metastatic colorectal cancer cells with high expression of GC-C are recognized through polypeptide targeting, release of cytotoxin is achieved through polypeptide conjugates (PDCs), it is further verified that the PDCs compound can achieve inhibition of the nM level of T84 cells, and the PDCs compound has good application prospects in preparation of drugs for treating colorectal tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a class of polypeptide conjugates, their preparation methods, and their application in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] According to reports, guanylate cyclase C (GC-C), which is highly expressed in metastatic colorectal cancer, has attracted widespread attention from medicinal chemists and pharmaceutical companies as a biomarker for this disease, and various forms of drugs based on it are in different stages of development. Currently, drug development targeting this marker is mainly divided into tumor suppression therapy with targeted delivery of cytotoxins and immunotherapy. Chemotherapy based on small molecule drugs (5-FU / LV, irinotecan, oxaliplatin, etc.) is one of the important means of treating metastatic colorectal cancer in tumor suppression therapy, but its cell killing effect generally lacks specificity, resulting in indiscriminate killing and causing significant side effects. Antibody-drug conjugates (ADCs) developed by utilizing the property of targeting GC-C to mediate and internalize to form endosomes, and exerting their effects through the lysosomal pathway, can overcome the limitations of poor specificity and low in vivo utilization of the aforementioned small molecule drugs to some extent. However, antibody-based macromolecules have certain limitations in cell internalization and tumor penetration, and the homogeneity of ADC drug products is not easy to guarantee. The certain differences in physicochemical properties between batches are a potential reason for the termination of existing ADC treatment regimens in clinical trials. Compared to ADCs, peptide delivery systems have higher tumor penetration. Peptide-drug conjugates (PDCs) that carry tumor cytotoxic peptides to exert antitumor effects have advantages such as simpler and more flexible design and synthesis, and easier achievement of stoichiometric drug conjugation. These advantages provide greater possibilities for developing highly efficient and low-toxicity conjugates and obtaining products with uniform modification and high homogeneity.

[0003] In the gut, a class of natural polypeptide ligands of GC-C bind to and activate downstream cGMP signaling pathways, thereby regulating specific processes in various key cell types, such as chloride and bicarbonate secretion, to maintain water and electrolyte balance inside and outside the intestine. Cyclic peptides developed based on these polypeptide ligands (such as linaclotide) have advantages such as high GC-C affinity and good stability in the digestive tract, and have been marketed as peptide drugs for the treatment of refractory constipation. However, there are currently no reports on using these GC-C-targeting peptide drugs to construct PDCs (proton pump inhibitors) targeting metastatic colorectal cancer for tumor-targeted therapy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application provides a class of PDC molecules that target metastatic colorectal cancer by constructing GC-C-targeting peptide drugs.

[0005] This invention first uses GC-C ligand peptides as carriers, and through precise modification, loads cytotoxins or target protein binding molecules. The peptides target and recognize metastatic colorectal cancer cells that highly express GC-C, and achieve the following through the GC-C-mediated endocytosis-lysosome pathway: by preparing peptide conjugates (PDCs), the cytotoxins are released in lysosomes, and cancer cells are inhibited by inhibiting intracellular targets.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a class of polypeptide conjugates having the following general structural formula: A-Linker-B, where A represents a functional molecule, including microtubule inhibitors such as TDM-1, MMAE, and MMAF; topoisomerase inhibitors such as SN38 and Dxd; RNA polymerase II inhibitors such as amatoxins; and other cytotoxic drugs that can directly kill tumor cells. Substances with similar functional molecule definitions and effects are all within the scope of protection claimed in this application, and will not be elaborated upon here.

[0008] Preferably, A is MMAE, a key intermediate for synthesizing MMAE, or a MMAE derivative.

[0009] B represents the target peptide, and A and B are connected by a linker, where B is either linaclotide or plecanatide. Linaclotide and plecanatide are marketed peptide drugs currently used to treat refractory constipation, with CAS registry numbers 851199-59-2 and 467426-54-6, respectively.

[0010] Furthermore, the GC-C targeting peptide used can be replaced by other types of linear or cyclic peptides with GC-C targeting function.

[0011] Furthermore, the linker of the peptide conjugate consists of three parts: a spacer, an enzyme-responsive portion, and a self-igniting portion. The spacer is used to regulate the distance between the target peptide and the functional molecule, as well as the physicochemical properties of the peptide conjugate molecule; it can be an adipose chain or a PEG chain. The enzyme-responsive portion is used to cleave the amide covalent coupling between the target peptide and the functional molecule; it can be a cathepsin B-cleavable linker, including a GGFG (Gly-Gly-Phe-Gly) tetrapeptide, a VC (Val-Cit) dipeptide, or a VA (Val-Ala) dipeptide. The self-igniting portion is used to release the functional molecule without leaving a trace after the linker is cleaved; it is composed of PAB (p-aminobenzyl alcohol). The linker is connected to the target peptide via amide condensation at the nitrogen or carbon terminus.

[0012] Furthermore, the linker structure is as follows:

[0013]

[0014] More specifically, the structural formula of the polypeptide conjugate is shown below:

[0015]

[0016]

[0017] Specifically, based on the above-mentioned polypeptide conjugates of the present invention, the effects of linker length, linker polarity, and linker site on the inhibitory effect on cell viability were investigated. Experiments revealed that differences in linker length, linker polarity, and linker site can all have unpredictable effects on cell inhibition.

[0018] In addition, the present invention also provides a method for preparing the above-mentioned polypeptide conjugate. This preparation method can also be carried out in combination with conventional techniques in the pharmaceutical field, and the present invention does not impose strict limitations on it.

[0019] This invention also provides a pharmaceutical composition comprising the aforementioned polypeptide conjugate as an active ingredient. Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, including excipients for oral formulations or parenteral administration. The route of administration can be oral, injection, topical, etc. According to the technical solution of this invention, the composition can be an oral or injectable formulation, and the excipients used include conventional excipients such as starch, sucrose, lactose, powdered sugar, glucose, mannitol, xylitol, polyethylene glycol, isopropanol, Tween-80, and glycerin.

[0020] Furthermore, this study found that Linaclotide itself did not exhibit cell viability inhibition of T84 cells. However, in the embodiments of this invention, multiple peptide conjugates—MMAE-L1-Linaclotide—were constructed using MMAE as a cytotoxic molecule. The peptide conjugate based on the GC-C targeting peptide was verified to inhibit metastatic colorectal cancer cells (T84 cells): the CCK-8 cell experiment verified the good inhibition of T84 cell viability by the peptide conjugate.

[0021] Therefore, the application of pharmaceutical compositions based on the aforementioned polypeptide conjugates and using polypeptide conjugates as active ingredients in the preparation of antitumor drugs is within the scope of protection claimed in this application. Furthermore, the tumor can be metastatic colorectal cancer.

[0022] definition

[0023] The term "ADC" is short for Antibody-Drug Conjugate, which is a small molecule drug with biological activity linked to a monoclonal antibody through a chemical chain. The term "PDC" is short for Peptide-Drug Conjugates. Compared with ADC drugs, PDC drugs have the characteristics of small molecular weight, strong tumor penetration and low immunogenicity.

[0024] The term "MMAE," short for Methylauratestatin E, is widely used in the development of antibody-drug conjugates (ADCs) for the treatment of various cancer types. The terms "key intermediates for the synthesis of MMAE" and "MMAE derivatives" refer to analogs that retain the cytotoxic function of MMAE itself.

[0025] The term "other cytotoxic drugs that can directly kill tumor cells" refers to other publicly disclosed cytotoxic substances used in PDC or ADC, other than microtubule inhibitors, topoisomerase inhibitors, or RNA polymerase II inhibitors as defined in existing studies.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] This invention starts with Linaclotide, a GC-C targeting peptide highly expressed in metastatic colorectal cancer. Through precise modification of this peptide, a peptide conjugate for tumor-targeted therapy against this disease is developed. This approach is expected to overcome the limitations of existing macromolecular-based treatments, such as the limitations of antibody-based macromolecules in cellular endocytosis and tumor penetration, as well as the difficulty in ensuring the homogeneity of antibody-drug conjugate products and the uncertainty of DAR values. In vitro experiments have verified that the synthesized peptide conjugate can achieve nM-level inhibition of T84 cells, demonstrating promising application prospects in the preparation of drugs for treating colorectal tumors. Attached Figure Description

[0028] Figure 1 Structural formula (A) and LC-MS spectrum (B) of compound N3-C5-PAB-MMAE;

[0029] Figure 2 Structural formula (A) and LC-MS spectrum (B) of compound N3-PEG4-PAB-MMAE;

[0030] Figure 3 Structural formula (A) and LC-MS spectrum (B) of compound MAL-C5-PAB-MMAE;

[0031] Figure 4 Schematic diagram (A) and LC-MS spectrum (B) of compound MMAE-GFGG-C5-(N)-Linaclotide;

[0032] Figure 5 Schematic diagram (A) and LC-MS spectrum (B) of compound MMAE-GFGG-PEG4-(N)-Linaclotide;

[0033] Figure 6 Schematic diagram (A) and LC-MS spectrum (B) of compound MMAE-GFGG-C5-(C)-Linaclotide;

[0034] Figure 7 Schematic diagram (A) and LC-MS spectrum (B) of compound MMAE-VC-C5-(N)-Linaclotide;

[0035] Figure 8 Cytotoxicity assay of the peptide conjugate MMAE-L1-Linaclotide against GC-C positive T84 cells. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and figures. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, preparation is carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are all conventional products that can be purchased commercially.

[0037] Preparation Examples: Synthesis of MMAE-L1-Linaclotide, a peptide conjugate targeting GC-C

[0038] 1. The synthesis of the cuttable connector + payload (L1+MMAE) unit is as follows:

[0039]

[0040] The specific preparation method is as follows:

[0041] Dissolve 20 mg of N3-C5-GGFG-PAB-PNP, 19 mg of MMAE, and 3.6 mg of HOBt in 3.5 mL of anhydrous DMF, add 8.3 μL of DIEA, and stir at room temperature. After reacting for 2 hours, the reaction progress is monitored using LCMS. The retention time of the target substance N3-C5-PAB-MMAE is t. R = 8.79 min. The reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain a white powder N3-C5-PAB-MMAE (28.8 mg, 95%).

[0042] LC-MS positive ion mode measurements yielded molecular weights of M+H = 1324.90 and M+Na = 1346.85, with theoretical molecular weights of M+H = 1324.77 and M+Na = 1346.75. Negative ion mode measurements yielded molecular weights of MH = 1322.75 and (M+HCOO). - =1369.50, theoretical molecular weight MH=1322.55, (M+HCOO) - =1368.76, see details for representation. Figure 1 The target product was synthesized using N3-PEG4-GGFG-PAB-PNP and MMAE as raw materials through similar synthetic steps.

[0043] N3-PEG4-GGFG-PAB-MMAE, characterization details can be found in [link to characterization documentation]. Figure 2 MAL-C5-VC-PAB-MMAE was synthesized using the cleavable linker MAL-C5-VC-PAB-PNP and MMAE as raw materials. For detailed characterization, see [link to characterization details]. Figure 3 .

[0044] 2. Synthesis of different types of polypeptide conjugates MMAE-L1-Linaclotide

[0045] 2.1 The synthesis of MMAE-L1-Linaclotide using a click reaction is shown below:

[0046]

[0047] The specific preparation method is as follows:

[0048] 4.5 mg of C3(N)-Linaclotide with an N-terminal alkynyl group and 3.2 mg of N3-C5-PAB-MMAE were dissolved in 0.8 mL of a 1 / 1 mixture of ACN and ammonium acetate (100 mM). 1 eq of CuSO4 was reduced with 2 eq VcNa, and the resulting solution was then added to the reaction system. The reaction progress was monitored using LCMS, and the retention time of the target product MMAE-GGFG-C5-(N)-Linaclotide was t. R =6.77 min. The reaction mixture was purified by preparative HPLC and lyophilized to give a white powder MMAE-GGFG-C5-(N)-Linaclotide (6.1 mg, 78%). The molecular weight was determined by LCMS to be (M+2H) / 2 = 1473.8.

[0049] (M+3H) / 3 = 983.1, theoretical molecular weight (M+2H) / 2 = 1473.6, (M+3H) / 3 = 983.0. For detailed characterization results, please refer to [link to characterization results]. Figure 4 .

[0050] 3. To investigate the effects of linker hydrophilicity and length on the activity of PDCs, a similar synthetic procedure was followed, using N3-PEG4-GGFG-PAB-MMAE and C3(N)-Linaclotide with an N-terminal alkynyl group as raw materials to synthesize MMAE-GGFG-PEG4-(N)-Linaclotide, which exhibits improved water solubility and can cleave linkers to elongate its length. Its structural schematic and LC-MS characterization results are shown below. Figure 5 To investigate the effect of different linkage sites on the activity of PDCs, the target product MMAE-GGFG-C5-(C)-Linaclotide was synthesized from C3-(C)-Linaclotide containing an alkyne group at the C-terminus and N3-C5-PAB-MMAE. Its structural schematic and LC-MS characterization results are shown below. Figure 6 .

[0051] 4. Synthesis of MMAE-L1-Linaclotide using thiol-Michael addition reaction

[0052]

[0053] To investigate the effect of different types of cleavable linkers on the PDCs of this invention, a peptide conjugate with dipeptide linkers, MMAE-VC-C5-(N)-Linaclotide, was prepared from MAL-C5-VC-PAB-MMAE and Cys-Linaclotide containing free cysteine ​​at the N-terminus, via a thiol-Michael addition reaction between free thiol groups and maleimide groups. Its structural schematic and LCMS characterization results are shown in [reference needed]. Figure 7.

[0054] The validation examples verified the inhibitory effect of the peptide conjugate on GC-C positive cells.

[0055] Two GC-C positive cell lines (T84 and HEK293-GC-C stable transgenic cells) were selected, with HEK293 as a negative control. The CCK-8 assay or SRB assay was used for detection. Cancer cells seeded in 96-well plates were treated with different concentrations of the peptide conjugate MMAE-GFGG-C6-Linaclotide and incubated for 72 hours. Subsequently, 10 μL of CCK-8 solution was added, and the plates were incubated for 1–4 hours. The absorbance (OD) at 450 nm was measured using a microplate reader.

[0056] Formula calculation: Cell viability (%) is calculated as follows: [A(drug-added) - A(blank)] / [A(0-drug-added) - A(blank)] × 100. Where A(drug-added): OD value of wells containing cells, CCK-8 solution, and drug solution; A(0-drug-added): OD value of wells containing cells and CCK-8 solution but no drug solution; A(blank): OD value of wells without cells.

[0057] The experiment also compared the effects of connector length, connector polarity, and connector sites on the inhibition of cell viability.

[0058] Experimental results are as follows Figure 8 As shown, before conjugation with the peptide, the standalone toxic molecule MMAE, due to its superior cell membrane penetration and strong cytotoxicity, already exhibited inhibitory effects on cell viability at the lowest concentration set in the experiment, serving as a positive control. The overall toxicity of the PDC molecules obtained after peptide conjugation was reduced; the cytoinhibitory ability of these PDC molecules was only demonstrated after they were endocytosed into lysosomes, cleaved by linker enzymes, and underwent electron transfer to release the intact MMAE. Detailed statistical data are shown in Table 1.

[0059] Table 1. Half-inhibitory concentration (WIC) of the peptide conjugate MMAE-L1-Linaclotide against T84 cells.

[0060]

[0061] According to the data in Table 1, MMAE-GFGG-C5-(N)-Linaclotide, with a tetrapeptide linker, showed the best inhibitory effect on T84 cell activity. The measured IC50 value was [missing data]. 50 =160.6 nM, which is superior to the cell inhibitory activity of MMAE-CV-C5-(N)-Linaclotide based on dipeptide linkers (the measured IC50 value is 160.6 nM). 50=1.537 μM). Low-polarity alkyl chain linkers (MMAE-GGFG-C5-(N)-Linaclotide, IC... 50 =160.6 nM) cell inhibitory activity was superior to that of the more polar linker (MMAE-GGFG-PEG4-(N)-Linaclotide, IC50). 50 =515.3 nM). The linker is MMAE-GGFG-C5-(C)-Linaclotide at the N-terminus of the peptide (the measured IC50 value is 515.3 nM). 50 =160.6 nM) cell inhibitory activity was superior to that of MMAE-GGFG-C5-(C)-Linaclotide (with the linker at the C-terminus of the peptide) (the tested IC50 was 160.6 nM). 50 =268.8 nM). Meanwhile, the control group of Linaclotide did not show any inhibitory effect on the cell viability of T84 cells.

[0062] The above embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A class of polypeptide conjugates, characterized in that, The polypeptide conjugate has the following general structural formula: A-Linker-B; Where A represents the functional molecule that constructs the ADC or PDC drug, and the functional molecule includes one or more of the following substances: Microtubule inhibitors: TDM-1, MMAE, or MMAF; Topoisomerase inhibitors: SN38 or Dxd; RNA polymerase II inhibitor: Amatoxins; Other cytotoxic drugs that can directly kill tumor cells; B represents the target peptide, and A and B are coupled through a linker, wherein B is lilonapeptide or pucanatide.

2. The polypeptide conjugate according to claim 1, characterized in that, The linker consists of three parts: a spacer, an enzyme-responsive portion, and a self-igniting portion; the spacer is an adipose chain or a PEG chain; the enzyme-responsive portion is a cathepsin B-cleavable linker, including a GGFG tetrapeptide, a VC dipeptide, or a VA dipeptide; the self-igniting portion is composed of PAB; the linker is connected to the target peptide B by amide condensation at the nitrogen or carbon terminus.

3. The polypeptide conjugate according to claim 1 or 2, characterized in that, The linker structure is one of the following structures:

4. The polypeptide conjugate according to claim 1 or 2, characterized in that, The polypeptide conjugate is one of the following structural formulas:

5. The polypeptide conjugate according to claim 1, characterized in that, A refers to MMAE, a key intermediate for the synthesis of MMAE, or an MMAE derivative.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the polypeptide conjugate of claim 1.

7. The use of the polypeptide conjugate of claim 1 or the pharmaceutical composition of claim 6 in the preparation of antitumor products.

8. The application according to claim 7, characterized in that, The tumor is metastatic colorectal cancer.

9. The application according to claim 7, characterized in that, The pharmaceutical composition is in the following formulations: powder, capsule, tablet, film, injection, or ointment.

10. The application according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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