Preparation and application of an anti-tumor peptide conjugate drug

Abstract

The present invention discloses an anti-tumor peptide conjugate drug and its application. The molecular sequence of the peptide conjugate drug is: MTX-Gly-Phe-Leu-Gly-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2, wherein the Gly-Phe-Leu-Gly sequence connected to MTX can be cleaved by cathepsin-B or the like. The anti-tumor peptide provided by the present invention has membrane permeability and can be used as a carrier for small molecule anti-tumor drugs to prepare peptide conjugate drugs, which can promote the anti-tumor drugs to penetrate the cell membranes of tumor cells and complete the release of small molecule drugs for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of protein polypeptide drugs, in particular to the field of anti-tumor drugs involved in the treatment of liver cancer, ovarian cancer and lymphoma, and specifically to the coupling of a polypeptide and an MTX drug and the application thereof. Background Art

[0002] Cancer has become a major global health concern and one of the leading causes of death worldwide. According to the World Health Organization, nearly 10 million people died from cancer in 2018. Furthermore, the number of new cancer cases worldwide is increasing at a rate of nearly 20 million per year. In 2020 alone, there were 19.3 million new cancer cases and nearly 10 million deaths.

[0003] There are many treatments for cancer, such as chemotherapy, radiotherapy, immunotherapy, and surgical resection. Among these treatments, surgical resection and radiotherapy are often used to treat non-metastatic cancer, while anticancer drugs (chemotherapy, hormones) are often used to treat metastatic cancer.

[0004] Small molecules have significant advantages in terms of metabolic stability and membrane permeability, but they also have higher off-target effects and therefore higher toxicity risks.

[0005] Compared with other small molecule drugs, peptide drugs have some unique advantages: the potential toxicity of peptide molecules is lower than that of small molecules, and they have relatively fewer off-target side effects.

[0006] Peptide-drug conjugates (PDCs) are a novel molecular drug delivery system, typically consisting of a peptide, a covalent linker, and a payload. The drug is typically bound to the peptide via a covalent linker, imparting bidirectional functionality to the peptide and drug, potentially enhancing the drug's killing or targeting effects. PDCs are a promising cancer treatment.

[0007] Melittin is a bioactive peptide extracted from the venom of European honey bees. It is a 26-amino acid cell-penetrating α-helical peptide. Melittin exhibits multi-target anti-tumor effects, including cell membrane disruption, anti-proliferation, anti-invasion, and anti-metastasis.

[0008] Methotrexate (MTX) is a drug commonly used to treat leukemia, but its further application is limited by its drug resistance. In addition, the low cellular uptake rate of MTX is a major challenge in MTX cancer treatment.

[0009] Covalent linkers come in a variety of types, including fatty chains, polyethylene glycol, and amino acids. Amino acids are one of the most commonly used covalent linkers in chemical synthesis. The GFLG tetrapeptide sequence has been widely used as a spacer that can be cleaved in the presence of cathepsin-B. The GFLG tetrapeptide spacer is most commonly used in molecular delivery systems, enabling site-specific cleavage of cathepsin-B in the tumor environment, thereby functioning as a covalent linker in molecular delivery systems.

[0010] To address the low cellular uptake of MTX, we designed a peptide-drug conjugate of MTX. MTX is linked to the honey bee toxin melittin via GFLG, creating a drug delivery system with the melittin peptide as the carrier and MTX as the payload. MTX and the glycine in the peptide are chemically synthesized via an amide bond, successfully constructing the MTX-GFLG-Melittin molecular delivery system.

[0011] Solid-phase peptide synthesis (SPPS) is currently the predominant method for synthesizing natural and non-natural peptides. Commonly used condensation systems for SPPS include HCTU / DIPEA, HATU / HOAt / DIPEA, and DIC / HOBt. These systems typically utilize a relatively low temperature of 30°C, resulting in a relatively long amino acid condensation reaction time.

[0012] Data show that the rapid synthesis of polypeptides can be achieved by using the DIC / Oxyma condensation system at 50°C. The advantages of using the DIC / Oxyma condensation system to complete the condensation reaction of amino acids at 50°C are as follows: (1) The synthesis speed of polypeptides is relatively fast, which can reduce the condensation time of amino acids by about 1 / 2. (2) The condensation of amino acids has strong robustness. (3) The racemization rate of amino acids in the condensation process is relatively low, and the racemization rate of amino acids in the condensation process is not higher than 3%. This method uses a high temperature method to synthesize the polypeptide part of the above-mentioned PDC drug. Summary of the Invention

[0013] To address the shortcomings of methotrexate in anti-tumor applications, especially the frequent use of methotrexate leading to drug resistance and low cellular uptake, which in turn leads to reduced anti-tumor efficacy, the present invention provides an anti-tumor polypeptide drug conjugate and its application.

[0014] The first aspect of the present invention provides a polypeptide conjugate drug, which comprises the anti-tumor polypeptide, a cytotoxic drug and a linker. Its molecular sequence is MTX-GFLG-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2

[0015] Furthermore, the cytotoxic drug is a small molecule tumor chemotherapy drug.

[0016] Preferably, the tumor chemotherapy drug is methotrexate.

[0017] Furthermore, the linker is a four-amino acid sequence Gly-Phe-Leu-Gly, GFLG. GFLG can be degraded by enzymes highly expressed in the tumor microenvironment and tumor cells, such as cathepsin-B.

[0018] Furthermore, the polypeptide-drug conjugate has a structure as shown in Formula A, wherein Melittin represents the anti-tumor polypeptide according to claim 1 and the peptide according to Formula A, and its amino acid sequence is H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2.

[0019]

[0020] The present invention provides a method for preparing the polypeptide-coupled drug, comprising the following steps:

[0021] (1) Melittin polypeptide chain was synthesized by DIC / Oxyma condensation system at 50℃. Fmoc solid phase synthesis method was used to couple Fmoc-Gln-OH to Rink-Amide AM resin, and then coupled with Fmoc-Gln-OH, Fmoc-Arg-OH, Fmoc-Lys-OH, Fmoc-Arg-OH, Fmoc-Lys-OH, Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-Ser-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Gly-OH in sequence to obtain a resin containing the Melittin polypeptide chain.

[0022] (2) After step (1) is completed, proceed to steps 2-a and 2-b.

[0023] Step 2-a: According to the solid phase synthesis method, the four amino acids in the linker are coupled to the Melittin polypeptide chain in the order of Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, and Fmoc-Gly-OH.

[0024] Step 2-b: According to the solid phase synthesis method, MTX is linked to a resin containing glycine via an amide bond to obtain a peptide-coupled drug resin, and the peptide is cleaved from the resin using a TFA cleavage solution, and then separated and purified to obtain the peptide-conjugate.

[0025] The second aspect of the present invention provides the use of the polypeptide-coupled drug in anti-tumor drugs.

[0026] Furthermore, the anti-tumor application is mainly to evaluate the killing effect on tumor cells including HepG2, Hepa1-6, ES-2, U937, Daudi, COC1 and A20 cells.

[0027] The beneficial effects of the present invention are:

[0028] 1. A rapid synthesis method for melittin polypeptide chains is provided. The DIC / Oxyma condensation system for synthesizing melittin polypeptide chains at 50°C offers the following advantages: rapid amino acid condensation, significantly reducing reaction time; low racemization rates during synthesis; and a robust, high-temperature resistant system.

[0029] 2. The anti-tumor polypeptide provided by the present invention has penetrability. The polypeptide of the present invention is used as a carrier of small molecule anti-tumor drugs to prepare polypeptide-coupled drugs. The anti-tumor polypeptide can penetrate the cell membrane of cancer cells, on the one hand, playing a role in killing tumor cells, and on the other hand, promoting the uptake of small molecule drugs at the tumor site, thereby increasing the therapeutic effect of the anti-tumor drug MTX.

[0030] 3. The polypeptide-conjugated drug of the present invention connects the bee toxin Melittin to methotrexate through a linker. The Gly-Phe-Leu-Gly tetrapeptide cleavage group can be degraded by cathepsin-B and other enzymes at the tumor site. It is an enzymatic linker and has strong specificity and high selectivity compared to traditional linkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis process of MTX-GFLG-Melittin peptide-drug conjugate;

[0032] Figure 2 Mass spectrometry (ESI-MS) analysis of pure bee toxin Melittin (MW: 2845.8);

[0033] Figure 3 Mass spectrometry (ESI-MS) analysis of pure peptide-conjugated drug A (MW: 3657.2);

[0034] Figure 4 Analytical reversed-phase high performance liquid chromatography (RP-HPLC) analysis spectra of crude and purified peptides of bee toxin Melittin;

[0035] Figure 5 Analytical reversed-phase high performance liquid chromatography (RP-HPLC) analysis spectra of crude and purified peptides of peptide-conjugated drug A;

[0036] Figure 6 The MTT method was used to detect the activity of MTX-GFLG-Melittin against adherent tumor cells (HepG2, Hepa1-6, and ES-2);

[0037] Figure 7 The CCK8 method was used to detect the activity of MTX-GFLG-Melittin against suspended tumor cells (U937, Daudi, COC1, and A20);

[0038] Figure 8 MTX activity against adherent tumor cells (HepG2, Hepa1-6, and ES-2);

[0039] Figure 9 MTX activity against suspended tumor cells (U937, Daudi, COC1, and A20); DETAILED DESCRIPTION

[0040] To more clearly understand the present invention, the present invention is further described with reference to the following examples. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0041] Example 1: Synthesis of polypeptide intrinsic sequence and linker ( Figure 1 ).

[0042] (1) Weighing of Rink-Amide AM resin

[0043] Remove the Rink-Amide AM resin from the refrigerator and allow it to cool to room temperature. Then weigh the Rink-Amide AM resin on a weighing balance. Weigh quickly to avoid moisture absorption. The resin weighs 600 mg and has a degree of substitution of 0.27 mmol / g.

[0044] (2) Activation of resin

[0045] The weighed resin was placed in a peptide synthesis tube, and 4 mL of DMF / DCM (v:v = 9:1) mixed reagent was added. The resin was activated at 30°C for more than 1 h to fully activate the resin.

[0046] (3) Standard washing of resin

[0047] Perform a standard wash on the activated resin. The standard wash procedure is as follows: First, wash the resin in the synthesis tube twice with DMF. Then, wash the resin twice with DCM. After that, wash the resin once again with DMF, and then once more with DCM. Finally, wash the resin three times with DMF.

[0048] (4) Removal of Fmoc protecting group on Rink-Amide AM resin

[0049] The Fmoc protecting group on the resin was removed using an Fmoc deprotection reagent (piperidine / DMF = 1:4, v:v). The Fmoc removal reaction was carried out at 30°C for two times, the first time for 5 minutes and the second time for 10 minutes.

[0050] (5) Preparation of Kaiser reagent and detection of Fmoc protecting group removal

[0051] Kaiser's reagent is a reagent for detecting amino groups. It consists of two main components. The preparation methods for both reagents are as follows: Reagent 1: Dissolve 1g of ninhydrin in 20mL of anhydrous ethanol. Reagent 2: Dissolve 16g of phenol in 4mL of anhydrous ethanol.

[0052] After the first Fmoc removal reaction, we perform a Kaiser test. The Kaiser test involves placing a small amount of Rink-Amide AM resin into a 200 μL centrifuge tube using a spotting capillary. Next, add one drop of ninhydrin solution and one drop of phenol solution to the tube in that order. After the solution is mixed thoroughly, heat it in a beaker for 1-2 minutes. After heating, remove the tube and observe the color of the resin. A color change indicates that the Fmoc protecting group has been removed.

[0053] (6) Activation of the first Fmoc-amino acid

[0054] Weigh 3eq of Fmoc-Gln-OH and 6eq of Oxyma racemization inhibitor into a 5mL centrifuge tube, then add pure DMF to dissolve the solid powder. Ultrasonic cleaning promotes the dissolution of the solid powder. Add 6eq of DIC compound to the dissolved solution and shake well. Then, activate it at room temperature for 3min-5min. Add the activated solution to the resin from which the Fmoc protecting group has been removed, and place the synthesis tube in a constant temperature shaker (50℃, 120 rpm) for reaction. The reaction is carried out twice, the first reaction for 15min-20min, and the second reaction for 20min-30min. The two reactions ensure the condensation reaction yield.

[0055] (7) Kaiser reagent to detect amino acid condensation

[0056] After the first amino acid condensation reaction is completed, we need to use Kaiser reagent to detect the first amino acid condensation reaction. The Kaiser test steps are as follows (5). Finally, observe the color of the resin. If the color of the resin turns colorless, the amino acid condensation reaction is successful.

[0057] (8) Extension of polypeptide sequence

[0058] The polypeptide chain was extended by repeating the following synthetic steps according to the above synthetic operation: Kaiser test, removal of the Fmoc protecting group, condensation of amino acids, Kaiser test, condensation of amino acids, and removal of the Fmoc protecting group. Fmoc solid-phase peptide synthesis technology is used to couple Fmoc-Gln-OH, Fmoc-Arg-OH, Fmoc-Lys-OH, Fmoc-Arg-OH, Fmoc-Lys-OH, Fmoc-Ile-OH, Fmoc-Trp-OH, Fmoc-Ser-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Thr-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, and Fmoc-Gly-OH were used to obtain the resin containing the Melittin polypeptide chain.

[0059] Arginine (Arg) is unstable at high temperatures, so the arginine condensation reaction was performed at 30°C. The amino acid condensation was performed using a molar ratio of Fmoc-arginine: HCTU: DIPEA = 3 eq: 2.8 eq: 6 eq. The arginine double condensation method is as follows: Weighed Fmoc-arginine and HCTU were placed in a 5 mL centrifuge tube. 4 mL of DMF solution was added to the tube and ultrasonic cleaning was performed to promote dissolution of the solids. Then, DIPEA solution was quickly added and the tube was immediately shaken. After adding DIPEA, the activated solution was added to the Fmoc-removed synthesis tube. Finally, the synthesis tube was placed in a thermostatic shaker (30°C, 120 rpm) for the reaction. The reaction was performed twice: the first reaction lasted 25-30 minutes, and the second reaction lasted 30-35 minutes. These two reactions ensured a high yield of the arginine condensation reaction.

[0060] (9) Synthesis of linkers

[0061] After the melittin polypeptide chain was coupled, the Fmoc group of the last amino acid, Fmoc-Gly-OH, on the resin was removed. Then, according to conventional methods for Fmoc solid-phase peptide synthesis, the four amino acids in the linker were coupled to the melittin polypeptide chain in the order of Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, and Fmoc-Gly-OH.

[0062] Example 2: Coupling of methotrexate and the polypeptide chain Melittin and its peptide cleavage.

[0063] Coupling of methotrexate and the melittin polypeptide chain. After removing the Fmoc protecting group from the last amino acid, the condensation reaction between MTX and the amino acid was carried out. The specific steps are as follows: 10 eq of methotrexate and 2 eq of HOBt were dissolved in DMF, followed by activation with 2 eq of DIC for 5 minutes. The reaction mixture was then added to a peptide synthesis tube and the reaction was repeated twice at 30°C for 1.5 hours each.

[0064] Peptide cleavage. Prepare 5 mL of TFA:TIPS:Phenol:H2O=88:2:5:5 (v:v:v:v) lysis solution in advance, add the pre-cooled lysis solution to the synthesis tube containing the resin, and react at 30°C for 2.5 hours. Filter, collect the filtrate using a three-necked flask, and then use high-purity N2 to bubble the filtrate and concentrate it to 4 mL. Add 40 mL of pre-cooled anhydrous ether to the three-necked flask and centrifuge to obtain a crude peptide. Finally, dissolve the crude peptide in acetonitrile / water, freeze it in a -80°C refrigerator, and freeze-dry the frozen crude peptide using a freeze dryer to obtain 101 mg of a crude product of a peptide-conjugated drug A. The structure of peptide-conjugated drug A is as follows:

[0065]

[0066] 101 mg of the crude product of the synthesized peptide-coupled drug A was separated and purified by semi-preparative RP-HPLC and dried in a freeze dryer to obtain a solid peptide pure product of the peptide-coupled drug A.

[0067] Example 3: Synthesis of crude Melittin and crude MTX-GFLG-Melittin, and analysis, separation and purification of crude Melittin and crude MTX-GFLG-Melittin.

[0068] The crude peptide sample was added with a small amount of acetonitrile and then added to deionized water to dissolve the crude peptide sample. The MTX-GFLG-Melittin crude peptide sample was sonicated using an ultrasonic cleaner to promote dissolution of the crude peptide sample. The resulting crude peptide solution was used for subsequent analysis, separation, and purification. Analytical RP-HPLC was used for analysis of the crude peptide, and semi-preparative RP-HPLC was used for separation and purification of the crude peptide.

[0069] Analytical RP-HPLC was performed using a C18 reverse-phase column (4.6 × 250 mm, 5-μm particle size) at a flow rate of 1 mL / min. Semi-preparative RP-HPLC was performed using a C18 reverse-phase column (10 × 250 mm, 10-μm particle size) at a flow rate of 4 mL / min. We monitored the entire analysis and purification process using wavelengths of 214 nm and 254 nm. For both analytical and semi-preparative RP-HPLC, the gradient of acetonitrile (containing 0.1% TFA) used for the analysis, separation, and purification of melittin was 20%-30% over 2 min, 30%-70% over 30 min. The gradient of acetonitrile (containing 0.1% TFA) used for the analysis, separation, and purification of MTX-GFLG-melittin was 30%-40% over 2 min, 40%-80% over 30 min.

[0070] Figure 4 and Figure 5 Analytical reversed-phase HPLC analysis of crude and purified peptides of melittin and MTX-GFLG-melittin, respectively. The figures clearly show that the purified melittin and MTX-GFLF-melittin contain significantly fewer impurities, resulting in higher-purity samples. Anti-tumor cell testing requires a sample purity greater than 95%.

[0071] The obtained solution was placed in a -80°C freezer and frozen overnight. Then, it was dried using a freeze dryer to obtain pure bee toxin melittin and pure MTX-GFLG-melittin in solid form.

[0072] The molecular weight of MTX-GFLG-Melittin was determined by electrospray tandem mass spectrometry (ESI-MS) and the mass spectra were recorded on a LTQ Orbitrap XL spectrometer (Thermo Scientific, USA).

[0073] Figure 2 and Figure 3 The following are the mass spectrometry (ESI-MS) analysis spectra of pure Melittin and MTX-GFLG-Melittin. The measured molecular weight of Melittin ([M+3H] 3+ =949.6) is 2845.8Da, and the theoretical molecular weight of Melittin is 2846.5Da. The measured molecular weight is consistent with the theoretical molecular weight, which indicates that the structure of the synthesized Melittin polypeptide is correct. 5+ =732.44) is 3657.2 Da, and the theoretical molecular weight of MTX-GFLG-Melittin is 3657.4 Da. The measured molecular weight is consistent with the theoretical molecular weight, which indicates that the structure of the synthesized MTX-GFLG-Melittin is correct.

[0074] Example 4: MTX-GFLG-Melittin anti-adherent tumor cell test.

[0075] (1) HepG2, Hepa1-6 and ES-2 adherent tumor cells were cultured at 10 4 Cells were seeded in 96-well plates at a volume of 100 μL / well and incubated overnight for 24 h.

[0076] (2) Add 50 μL of DMEM complete medium containing MTX-GFLG-Melittin at concentrations of 0.3, 0.9, 3, 9, 30, and 90 μM to a 96-well plate and incubate in a cell culture incubator for four hours.

[0077] (3) Add 15 μL of 5 mg / L MTT solution and incubate in a cell culture incubator for 4 h.

[0078] (4) Aspirate the cell culture medium in the 96-well plate, add 150 μL of DMSO to each well, and incubate in a cell culture incubator for 1 hour.

[0079] (5) Shake the 96-well plate and record the absorbance at 492 nm.

[0080] (6) The control group did not receive any drug, and the zero-adjustment group received a culture medium without cells, and the operation was the same as that of the experimental group.

[0081] (7) Anti-adherent tumor cell activity = (A492nm control - A492nm test) / (A492nm control - A492nm zero adjustment).

[0082] The anti-adherent tumor cell activity of MTX-GFLG-Melittin was calculated. The activity data were expressed as mean ± SEM. The cell experiment was repeated three times (n = 3). The activity data were statistically analyzed using SPSS software. The statistical results are shown in Table 1.

[0083] Example 5: MTX anti-adherent tumor cell test.

[0084] (1) HepG2, Hepa1-6 and ES-2 adherent tumor cells were cultured at 10 4 Cells were seeded in 96-well plates at a volume of 100 μL / well and incubated overnight for 24 h.

[0085] (2) Add 50 μL of DMEM complete medium containing MTX at concentrations of 3, 9, 30, 90, 30, and 900 μM to a 96-well plate and incubate in a cell culture incubator for four hours.

[0086] (3) Add 15 μL of 5 mg / L MTT solution and incubate in a cell culture incubator for 4 h.

[0087] (4) Aspirate the cell culture medium in the 96-well plate, add 150 μL of DMSO to each well, and incubate in a cell culture incubator for 1 hour.

[0088] (5) Shake the 96-well plate and record the absorbance at 492 nm.

[0089] (6) The control group did not receive any drug, and the zero-adjustment group received a culture medium without cells, and the operation was the same as that of the experimental group.

[0090] (7) Anti-adherent tumor cell activity = (A492nm control - A492nm test) / (A492nm control - A492nm zero adjustment).

[0091] The anti-adherent tumor cell activity of MTX was calculated. The activity data were expressed as mean ± SEM. The cell experiment was repeated three times (n = 3). The activity data were statistically analyzed using SPSS software. The statistical results are shown in Table 1.

[0092] Table 1: Antitumor activity of different drugs against three adherent tumor cells

[0093]

[0094] The statistical results of MTX and MTX-GFLG against adherent tumor cells (HepG2, Hepa1-6 and ES-2) are shown in Table 1. Figure 6 and Figure 8 As shown. For three adherent tumor cells, the IC 50 All of them are greater than 300μM, which indicates that the anti-adherent tumor effect of MTX is not ideal. For MTX-GFLG-Melittin drug, its IC 50 The concentrations of MTX-GFLG-Melittin were all less than 5 μM, indicating that MTX-GFLG-Melittin possesses strong anti-adherent tumor cell activity. Compared to MTX alone, the anti-adherent tumor cell activity of MTX-GFLG-Melittin was several dozen times greater. These results demonstrate that melittin, as a lead compound, can enhance the anti-adherent tumor cell activity of MTX.

[0095] Example 6: MTX-GFLG-Melittin anti-suspended tumor cell test.

[0096] (1) U937, Daudi, COC1 and A20 suspended tumor cells were cultured at 10 4 Cells were seeded in 96-well plates at a volume of 100 μL / well and incubated overnight for 24 h.

[0097] (2) Add 50 μL of DMEM complete medium containing MTX-GFLG-Melittin at concentrations of 0.3, 0.9, 3, 9, 30, and 90 μM to a 96-well plate and incubate in a cell culture incubator for four hours.

[0098] (3) After incubation, 15 μL of CCK8 working solution was added to each well, and the cells were incubated in a cell culture incubator for 3 hours. The absorbance was measured at 450 nm using a microplate reader.

[0099] (4) Anti-suspended tumor cell activity = (A450nm control - A450nm experimental) / (A450nm control - A450nm zero adjustment).

[0100] (5) The anti-adherent tumor cell activity of MTX-GFLG-Melittin was calculated. The activity data were expressed as mean ± SEM. The cell experiment was repeated three times (n = 3). The activity data were statistically analyzed using SPSS software. The statistical results are shown in Table 2.

[0101] Example 7: MTX anti-suspended tumor cell test.

[0102] (1) U937, Daudi, COC1 and A20 suspended tumor cells were cultured at 10 4 Cells were seeded in 96-well plates at a volume of 100 μL / well and incubated overnight for 24 h.

[0103] (2) Add 50 μL of DMEM complete medium containing MTX at concentrations of 3, 9, 30, 90, 300, and 900 μM to a 96-well plate and incubate in a cell culture incubator for four hours.

[0104] (3) After incubation, 15 μL of CCK8 working solution was added to each well, and the cells were incubated in a cell culture incubator for 3 hours. The absorbance was measured at 450 nm using a microplate reader.

[0105] (4) Anti-suspended tumor cell activity = (A450nm control - A450nm experimental) / (A450nm control - A450nm zero adjustment).

[0106] (5) The anti-adherent tumor cell activity of MTX was calculated. The activity data were expressed as mean ± SEM. The cell experiment was repeated three times (n = 3). The activity data were statistically analyzed using SPSS software. The statistical results are shown in Table 2.

[0107] Table 2: Antitumor activity of different drugs against four types of suspended tumor cells

[0108]

[0109] The statistical results of MTX and MTX-GFLG-Melittin against suspended tumor cells (U937, Daudi, COC1 and A20) are shown in Table 2. Figure 7 and Figure 9 As shown. For a single MXT, its IC 50 greater than 300 μM, while the IC 50The results were all below 3μM. In comparison, the anti-suspended tumor cell effect of MTX-GFLG-Melittin was increased by at least 100 times. This shows that using Melittin peptide as a lead compound can effectively enhance the anti-suspended tumor effect of MTX.

[0110] These anti-tumor activity results demonstrate that MTX-GFLG-Melittin exhibits potent cytotoxicity against a wide range of tumor cell types. This confirms the broad-spectrum anti-tumor potential of MTX-GFLG-Melittin. Most importantly, compared to the anti-tumor effect of MTX alone, MTX-GFLG-Melittin significantly enhances its cytotoxicity against seven tumor cell types, providing insights and insights for the subsequent development of PDC anti-tumor drugs.

[0111] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any slight modification of the contents of the present invention, or direct or indirect application in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of a peptide-coupled drug in the preparation of an anti-liver cancer drug, characterized in that The structural formula of the polypeptide-coupled drug is shown in Formula A. The peptide in formula A is bee toxin Melittin, and its amino acid sequence is: Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln.

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