CN116199747A - Lidamycin prodrug, preparation method and application thereof in the preparation of antitumor drugs - Google Patents

Abstract

The invention relates to a lidamycin prodrug, a preparation method and application thereof in preparing antitumor drugs, wherein a conjugate of lidamycin and maleimide is creatively prepared, and the drug can be quickly combined with free sulfhydryl of albumin after entering the body, so that the lidamycin is targeted and delivered to a tumor part to play an antitumor role. At present, no report on the preparation of the lidamycin prodrug by a chemical method and the application of the lidamycin prodrug as a targeted antitumor drug exists, the preparation method is simple, and the lidamycin prodrug can be prepared by only one-step reaction.

Description

Lidamycin prodrug, preparation method and its use in the preparation of antitumor drugs application

technical field

The invention belongs to the technical field of medicine, and more specifically, the invention relates to a lidamycin prodrug, a preparation method and its application in the preparation of antitumor drugs.

Background technique

Lidamycin (LDM, formerly known as C-1027) is produced by an actinomycete (Streptomyces globisporus C-1027, strain preservation number: CGMCC No.0704) isolated from the soil of Qianjiang County, Hubei Province. macromolecular peptide antibiotics. Lidamycin is composed of a 110-amino acid prosthetic protein (lidaprotein, LDP) and an active enediyne chromophore (active enediyne, AE), the two are non-covalently bonded, and can be disassembled and rebuilt . The chromophore is the active part of lidamycin, but it is unstable; the prosthetic protein protects the chromophore. Lidamycin has a strong killing effect on a variety of tumor cells in vitro, and has a significant effect on transplanted mouse colon cancer, human liver cancer and cecum cancer in vivo (Chinese Journal of Antibiotics, 1994, 19: 164-8). Mechanistic studies have shown that lidamycin can cause specific DNA breaks, thereby inducing apoptosis. Due to its strong cytotoxicity, lidamycin is a highly effective "warhead" drug for the preparation of targeted drugs.

At present, our laboratory has prepared a variety of genetically engineered lidamycin-targeted fusion proteins (Clin Cancer Res, 2010, 16:2085-2094). These drugs are mainly prepared by DNA recombination technology to produce fusion proteins of LDP and targeting molecules, and then Prepared by assembling chromophore AE through molecular strengthening technology. The preparation process of this method system is complex, involving the construction of expression vectors, transfection, fermentation, expression, purification, denaturation, renaturation of fusion proteins, chromophore extraction, assembly and other steps; and the assembly efficiency of prosthetic protein and chromophore Limited, after optimizing various conditions, the assembly efficiency can only reach 67% (Biomed EnvironSci, 2011, 24:602-607).

In addition, free chromophores extracted from LDM are not protected by LDP and are more prone to inactivation. The above reasons lead to inhomogeneous final products, containing fusion proteins with unassembled chromophores, fusion proteins with active chromophores, and fusion proteins with inactive chromophores. The existence of inactive products not only reduces the activity of the final product, but also competes with the fusion protein of the assembled AE for the targeting binding site, reducing the tumor targeting of the drug. The laboratory has also used chemical methods to prepare anti-type IV collagenase monoclonal antibody 3G11 and its Fab' fragments and chemical immunoconjugates of LDM (patent numbers: CN200510066529.4, CN02153655.4), anti-type IV collagenase monoclonal antibody The chemical immunoconjugate of 3D6 and LDM (patent application number: CN02125314.5). However, the antibodies 3G11 and 3D6 selected for the above-mentioned immunoconjugates are mouse-derived monoclonal antibodies, and human anti-mouse antibody reactions will occur when used in humans, and the above-mentioned immunoconjugates are mainly suitable for colonic cells with high expression of type IV collagenase. cancer, liver cancer, etc.

Albumin (albumin) is a heart-shaped protein with a molecular weight of about 67kDa, which is extremely abundant in human plasma, as high as 35-50mg/mL. In addition to being an important nutrient, albumin also plays an important role in maintaining plasma colloid osmotic pressure and reducing the toxicity of heavy metal ions. Albumin is a non-specific transport protein, which contains multiple pocket structures formed by hydrophobic and positively charged groups, and can form soluble complexes with poorly soluble substances. Good transport ability coupled with its non-immunogenic, biocompatible and biodegradable properties make it an excellent vehicle for drug delivery. In tumor therapy, albumin can be used for targeted delivery of antitumor drugs. On the one hand, albumin receptors (glycoprotein Gp60 and acidic secretory protein SPARC) are expressed on the surface of vascular endothelial cells and most tumor cells, which can realize the active targeting of drugs; on the other hand, due to the high permeability of tumor blood vessels In the absence of sex and lymphatic reflux, albumin molecules can be specifically enriched at tumor sites to achieve passive targeted delivery of drugs. Studies have shown that albumin contains a total of 35 cysteines (Cys), forming 17 pairs of disulfide bonds and 1 unpaired free sulfhydryl group (Cys34).

At present, albumin has been reported as a prodrug strategy for improving tumor chemotherapy (patent number: CN201080018619.5). The strategy uses a degradable spacer arm to link the growth inhibitor to albumin, which is cleaved by the prostate-specific antigen at the tumor site to release the drug. However, the use of degradable spacer arms will increase the risk of off-target, and it is only suitable for specific cancers with high expression of prostate specific antigen, such as prostate cancer. In addition, various reactions of the strategy must be carried out in organic solvents. This reaction system will lead to the denaturation of LDP and the loss of chromophores, reducing the activity of LDM, so it is not suitable for the preparation of LDM prodrugs.

At present, there is no relevant report on the preparation of lidamycin prodrug by chemical method and as a targeted anti-tumor drug. The object of the present invention is to provide a lidamycin prodrug capable of binding to albumin, its preparation method and its application in tumor targeting therapy.

Contents of the invention

For above technical problem, the first aspect of the present invention provides a kind of lidamycin prodrug, its chemical formula is as shown in formula 1:

M-L-LDM formula 1

Wherein, M represents maleimide; LDM represents lidamycin; L is a spacer arm, which is selected from carbonyl, C _1-10 carbonyl, C ₂ (PEG) _2-24 carbonyl, carbonyl substituted methyl cycloalkane Any of the groups, preferably methylcyclohexanecarboxamido, phenyl, propionamide, carbonyl-substituted methylcyclopropanyl, carbonyl-substituted methylcyclobutanyl, carbonyl-substituted methyl Cyclopentyl, carbonyl-substituted methylcyclohexyl, carbonyl-substituted methylcyclohexyl, carbonyl-substituted methylcyclooctyl, carbonyl-substituted methylcyclohexyl is more preferred. Among them, M, L and LDM are preferably combined through covalent bonds, but the present invention does not specifically limit this.

A further lidamycin prodrug, which consists of lidamycin and a maleimide group covalently bound to it, has a chemical formula as shown in formula 2:

Mal-LDM Formula 2

Wherein, Mal represents a maleimide group, which includes a maleimide and a spacer arm; LDM represents lidamycin. In addition, the connection between Mal and LDM is preferably through a covalent bond, but the present invention does not specifically limit this.

Further lidamycin prodrug, described maleimide group is maleimide methyl cyclohexane group, maleimide methyl cyclohexane carboxamide group, maleimide phenyl, maleimidepropionamide, maleimidemethylcyclopropanyl, maleimidemethylcyclobutanyl, maleimidemethylcyclopentyl, maleimide Imidomethylcycloheptyl, maleimidomethylcyclooctyl; preferably, maleimidomethylcyclohexyl is 4-(N-maleimidomethyl ) cyclohexyl group.

As a further lidamycin prodrug, the 1st position of the aliphatic ring of the 4-(N-maleimidomethyl)cyclohexane group is connected with the prosthetic group protein of the lidamycin through the carbonyl group. The amino groups are connected, and the 3rd position of the five-membered heterocycle of the 4-(N-maleimidomethyl)cyclohexane group is combined with the sulfhydryl group of albumin.

The second aspect of the present invention provides the preparation method of lidamycin prodrug, this preparation method comprises at least one end containing the crosslinking agent of maleimide group and lidamycin reaction step; Preferably, crosslinking The other end of the agent contains a succinimide group or a sulfosuccinimide group, respectively. Further preferred crosslinking agents are selected from the following compounds: succinimide 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and Sulfo-SMCC, succinimide 4 -(N-maleimidomethyl)cyclohexane-1-carboxy-(6-aminocaproate) (LC-SMCC); PEGylated SMCC crosslinker SM(PEG) _2-24 ; Succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) and Sulfo-SMPB, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS) and Sulfo-MBS; and succinimidyl-6-maleimidopropionamidohexanoate (SMPH); and N-alpha-maleimidoacetyl-oxysuccinimide Amino ester (AMAS), N-β-maleimidopropionyl-oxysuccinimide ester (BMPS), N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS ) and Sulfo-GMBS, N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) and Sulfo-EMCS, N-κ-maleimidoundecanoyloxy-sulfo Succinimidyl ester sodium salt (Sulfo-KMUS).

In the further preparation method of lidamycin prodrug, the maleimide and succinimide ester groups of the cross-linking agent are spacer arms, which are selected from carbonyl, C _1-10 carbonyl, C ₂ ( PEG) Any of _2-24 carbonyl, carbonyl-substituted methylcycloalkyl, preferably methylcyclohexanecarboxamido, phenyl, propionyl, carbonyl-substituted methylcyclopropanyl, carbonyl Substituted methylcyclobutanyl, carbonyl substituted methylcyclopentyl, carbonyl substituted methylcyclohexyl, carbonyl substituted methylcycloheptyl, carbonyl substituted methylcyclooctyl, further Preferred is carbonyl-substituted methylcyclohexyl.

In a further method for preparing lidamycin prodrug, the cross-linking agent is Sulfo-SMCC.

The further preparation method of lidamycin prodrug is: take appropriate amount of LDM and dissolve in PBS-ETDA solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ 12H ₂ O: 2.9g/L, NaCl: 8.0 g/L, KCl: 0.2g/L, 0.58g/L ethylenediaminetetraacetic acid, pH 7.2), add Sulfo-SMCC dissolved in pure water, wherein the molar ratio of LDM to Sulfo-SMCC is 1:0.1～1000, The preferred ratio is 1:1-100. The lidamycin prodrug can be prepared by reacting at 4-37° C. in the dark for 1 minute to 48 hours, preferably at room temperature in the dark for 10-120 minutes.

A further preparation method of lidamycin prodrug, the preparation method also includes the separation and purification step of removing small molecules by gel filtration chromatography or ultrafiltration.

The third aspect of the present invention also provides the application of lidamycin prodrug in the preparation of antitumor drugs.

The fourth aspect of the present invention also provides a preformed conjugate of lidamycin prodrug and albumin, its preparation method and application. The preparation method of the preformed conjugate is as follows: mix the purified LDM prodrug and albumin at a molar ratio of 1:0.1 to 200, preferably 1:0.2 to 10, and react at 4 to 37°C for 1 minute to 48 hours in the dark , preferably at 37° C. for 10 to 120 minutes in the dark, to prepare a preformed lidamycin-albumin conjugate.

The advantages and positive effects of the present invention are that, as an anti-tumor targeted drug, the lidamycin prodrug can quickly combine with albumin after entering the body, and shows good tumor targeting. It shows stronger anti-tumor activity and lower system toxicity; the preparation method is simple, and only one step reaction can prepare lidamycin prodrug, compared with the lidamycin strengthened fusion protein prepared by genetic engineering method , the preparation process is simple, no need to express protein and reassemble chromophore; low immunogenicity, it exerts targeting by combining with endogenous albumin, and there is no human anti-mouse antibody reaction; lidamycin and albumin pass through Non-degradable spacer arm connection, low off-target risk; broad anti-tumor spectrum, using albumin as the transport carrier, can target LDM to a variety of tumors. Therefore, the lidamycin prodrug of the present invention has the characteristics of simple preparation process, good targeting, low immunogenicity, low off-target risk, and broad antitumor spectrum, and has good clinical application prospects.

Description of drawings

Figure 1 MCC-rLDP molecular weight identification;

Figure 2 rLDP molecular weight identification;

The binding of Fig. 3rLDP prodrug to albumin;

Among them: M-protein molecular weight standard; 1-rLDP; 2-HSA; 3-MCC-HSA; 4-M(PEG) ₂ -HSA;

Fig. 4 The binding site between MCC-rLDP and albumin;

Among them: M-protein molecular weight standard; 1-rLDP; 2-MCC-rLDP; 3-HSA; 4-MCC-rLDP+HSA; 5-MCC-rLDP+HSA+Thiol blocked;

Figure 5 electrophoresis detection of the combination of MCC-rLDP and albumin;

Among them: M- protein molecular weight standard;

The binding kinetics of Fig. 6MCC-rLDP and albumin;

The distribution of Figure 7MCC-rLDP-Cy7 in mice;

Among them: 1-0.5 hours; 2-6 hours; 3-24 hours; 4-2 days; 5-3 days; 6-4 days; 7-5 days; 8-7 days;

Figure 8 The distribution of rLDP-Cy7 in mice;

Among them: 1-0.5 hours; 2-6 hours; 3-24 hours; 4-2 days; 5-3 days;

The in vivo antitumor activity of Fig. 9MCC-LDM and LDM;

The influence of Fig. 10MCC-LDM and LDM on mouse body weight;

Figure 11 The weight loss rate of mice in MCC-LDM and LDM groups at the end of the experiment.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

In some embodiments of one aspect of the present invention, the conjugates of lidamycin and maleimide groups are creatively prepared. After the drug enters the body, it can quickly combine with the free sulfhydryl group of albumin, so that lidamycin Targeted delivery of the hormone to the tumor site, releasing the chromophore AE non-covalently combined with LDP, exerting anti-tumor effect.

Some embodiments of another aspect of the present invention also creatively provide a preparation method of lidamycin prodrug. The chemically modified technical route uses a maleimide-containing cross-linking agent to react with lidamycin, and then uses gel filtration chromatography or ultrafiltration to purify the product.

According to some preferred embodiments of the present invention, the selected cross-linking agent contains a succinimide ester group and a maleimide group, and the succinimide ester group can react with the amino group on the LDP to generate lidamycin Prodrug (Mal-LDM); Mal-LDM contains a maleimide group, which can react with free sulfhydryl groups on albumin (human serum albumin, HSA) to generate a conjugate of lidamycin and albumin ( LDM-HSA), the reaction process is as follows:

According to some other preferred embodiments of the present invention, the present invention also provides a preparation method of recombinant lidamycin prosthetic protein (rLDP) prodrug, and the preparation strategy is the same as that of lidamycin prodrug. The reason for preparing the rLDP prodrug is that rLDP contains a His-tag tag, which is convenient for subsequent purification and identification research; in addition, LDM has strong cytotoxic activity, while rLDP has no obvious cytotoxic activity, which is convenient for in vivo imaging research.

According to some other preferred embodiments of the present invention, the present invention also provides the activity test research of the lidamycin prosthetic protein prodrug and lidamycin prosthetic protein prodrug, including lidamycin prosthetic protein prodrug and white Analysis of protein binding activity, binding kinetics of lidamycin prosthetic group protein prodrug and albumin, detection of cytotoxicity of lidamycin prodrug and preformed conjugates on tumor cells, detection of lidamycin prosthetic group The distribution of protein prodrugs in tumor-bearing mice, and the animal experimental treatment scheme of lidamycin prodrugs.

The following examples further illustrate the invention.

The preparation of embodiment 1 lidamycin and lidamycin prosthetic protein prodrug

This embodiment selects Sulfo-SMCC (CAS: 92921-24-9, formula 3, Shanghai Bi De Pharmaceutical Technology Co., Ltd.) and SM (PEG) ₂ (CAS: 955094-26-5, formula 4, Shanghai Bi De Medicine Technology Co., Ltd.) as a cross-linking agent to prepare lidamycin prodrug and recombinant lidamycin prosthetic protein (rLDP) prodrug. The preparation method of rLDP can be found in literature (Chinese Journal of Antibiotics, 2010, 4:265-269).

1. Dissolve 0.2mg of LDM in 1mL of PBS-ETDA solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ 12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L L, 0.58g/L ethylenediaminetetraacetic acid, pH 7.2), add 0.25mg of Sulfo-SMCC dissolved in pure water, react at room temperature in the dark for 30 minutes to prepare the prodrug MCC-LDM.

2. Dissolve 1.17mg rLDP in PBS-ETDA solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ 12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L , 0.58g/L ethylenediaminetetraacetic acid, pH 7.2), add 0.5mg of Sulfo-SMCC dissolved in pure water, and react at room temperature for 30 minutes to prepare the prodrug MCC-rLDP.

3. Dissolve 0.2mg of LDM in PBS-ETDA solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ 12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L , 0.58g/L ethylenediaminetetraacetic acid, pH 7.2), add 0.25mg of SM(PEG) ₂ dissolved in an organic solvent such as dimethyl sulfoxide, and react in the dark for 30 minutes at room temperature to prepare the prodrug M(PEG ) ₂ -LDM.

4. Dissolve 1.17mg rLDP in PBS-ETDA solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ 12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L , 0.58g/L ethylenediaminetetraacetic acid, pH 7.2), add 0.5mg of SM(PEG) ₂ dissolved in an organic solvent such as dimethyl sulfoxide, and react at room temperature for 30 minutes to prepare the prodrug M(PEG) ₂ - rLDP.

In the present invention, MCC-LDM refers to the prodrug prepared by Sulfo-SMCC and LDM, M(PEG) ₂ -LDM refers to the prodrug prepared by SM(PEG) ₂ and LDM, and MCC-rLDP refers to Sulfo-SMCC and rLDP The prepared prodrug, M(PEG) ₂ -rLDP refers to the prodrug prepared by SM(PEG) ₂ and rLDP.

Example 2. Purification of lidamycin and lidamycin prosthetic protein prodrug

In this example, a PD-10 column (GE Healthcare) was used to purify the product. The PD-10 column used PBS (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ ·12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L, pH 7.2) wash the column 5 times, the product of Example 1 is fixed to 2.5mL loading sample, discard the eluate, add 3.5mL PBS to collect the eluate and it is end product. When the volume of the product prepared in Example 1 is relatively large, use an ultrafiltration tube (Millipore) with a cut-off of 3 kDa to centrifuge at 5000 g for 30 minutes, and repeat 3 times to remove small molecules.

Example 3. Mass spectrometry identification of lidamycin prosthetic protein prodrug

The final product MCC-rLDP and rLDP purified after the reaction of Sulfo-SMCC and rLDP were used to detect the molecular weight by LC-MS. Take an appropriate amount of solution sample, centrifuge at 12,000 rcf for 10 minutes at 4°C, and collect the supernatant into a new centrifuge tube for later use.

Instruments used: Capillary high-performance liquid chromatography Ultimate 3000 (Thermo Fisher Scientific, USA), electrospray-quadrupole time-of-flight mass spectrometer AB SCIEX TripleTOF 5600Mass Spectrometer (ABSCIEX, USA)

1.7μm,2.1mm×50mm)；2)流动相A：0.1％甲酸,H₂O；3)流动相B：0.1％甲酸,乙腈；4)流速：0.3mL/分钟；5)液相色谱梯度：15分钟；洗脱条件如下：Ultra-high performance liquid chromatography conditions: 1) chromatographic column: ACQUITY UPLC Protein BEH C4Column (

1.7μm, 2.1mm×50mm); 2) Mobile phase A: 0.1% formic acid, H ₂ O; 3) Mobile phase B: 0.1% formic acid, acetonitrile; 4) Flow rate: 0.3mL/min; 5) Liquid chromatography gradient : 15 minutes; the elution conditions are as follows:

time (minutes) Solvent B(%) 0 5 10 100 13 100 14 5 15 5

Mass spectrometry conditions: 1) Scan Range: m/z 600–6000; 2) Output Mass: M; 3) S/N Threshold: 3; 4) Rel. Abundance Threshold (%): 0; 5) Charge Range: 5- 50; 6) Min. Num Detected Charge: 2; 7) Isotope Table: Protein.

The data analysis of the measurement results showed that the molecular weight of MCC-rLDP was 11638.5Da (Figure 1), and the molecular weight of rLDP was 11419.1Da (Figure 2), with a difference of 219.4, indicating that a molecule of maleimide group was successfully integrated into rLDP.

Example 4. Binding activity of lidamycin prosthetic protein prodrug to albumin

The two rLDP prodrugs purified in Example 2 were mixed with human serum albumin HSA (Sigma) at a molar ratio of 1:2, reacted at 37°C for 30 minutes, and the reaction product was stained with Coomassie brilliant blue after polyacrylamide gel electrophoresis , to detect the target band. Figure 3 shows that compared with HSA, after the two prodrugs react with HSA, the target bands can be seen around 75kDa, indicating that both prodrugs can combine with HSA to generate rLDP-HSA conjugates. To detect whether the binding site of rLDP prodrug to HSA is HSA-Cys34, the sulfhydryl blocker 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid was used (CAS: 57078-98-5, Shanghai Pide Pharmaceutical Technology Co., Ltd.) to block the Cys34 site of HSA, and then react with MCC-rLDP. Figure 4 shows that the target band does not appear around 75kDa after adding the sulfhydryl blocking agent, indicating that HSA-Cys34 is the binding site of MCC-rLDP.

Example 5. Binding kinetics of lidamycin prosthetic protein prodrug and albumin

The purified MCC-rLDP and human serum albumin were respectively dissolved in PBS-ETDA solution, preheated at 37°C for 10 minutes, and mixed at a molar ratio of 1:2, at 0, 1, 2, 4, 8, 16 , Take samples at 32 and 64 minutes, add 100-fold excess sulfhydryl blocking agent to terminate the reaction, and stain with Coomassie Brilliant Blue after polyacrylamide gel electrophoresis to detect product bands. The results are shown in Figure 5, MCC-rLDP rapidly combined with HSA. Grayscale analysis was performed on the MCC-rLDP band shown in Figure 5 and the binding rate was calculated. The results are shown in Figure 6. After 32 minutes of reaction, the binding rate of MCC-rLDP to HSA was about 81.5%.

Example 6. Preparation and purification of preformed conjugates

Dissolve MCC-LDM and human serum albumin in PBS-ETDA solution at a molar ratio of 2:1, react in the dark at 37°C for 30 minutes, and centrifuge at 5000g for 30 minutes using an ultrafiltration tube (Millipore) with a cutoff of 30kDa, repeat 3 times Once, the unreacted MCC-LDM is removed to prepare the preformed lidamycin-albumin conjugate LDM-HSA.

Example 7. Cytotoxic activity of lidamycin prodrugs and preformed conjugates on tumor cells

The cytotoxic activity of lidamycin prodrug, preformed conjugate LDM-HSA and lidamycin was detected by MTT (tetramethyl azolium salt) method. Human large cell lung cancer NCI-H460 cells, human lung cancer A549 cells, human squamous cell carcinoma KB cells and human liver cancer SMMC-7721 cells were treated with 10% fetal bovine serum (Gibco BRL Inc.), 2mM glutamine, 100μg/mL streptomycin and RPMI-1640 medium (Gibco BRL Inc.) of 100U/mL penicillin were cultivated in an incubator (ThermoScientific, 3131 carbon dioxide incubator) containing 5% _CO2 at 37°C. The above tumor cell lines are all common cell lines, which are preserved in our laboratory, and can also be purchased from commercial channels such as ATCC cell bank (Rockville, MD, USA) and the National Experimental Cell Resource Sharing Platform. The cells in the logarithmic growth phase were digested and counted, and 4000 cells/well were spread on a 96-well plate. After culturing for 24 hours, different concentrations of drugs were added, and three parallel wells were set for each drug concentration. After continuing to culture for 48 hours, 20 μL of MTT (Beiyuntian) dissolved in PBS at 5 mg/mL was added to each well. After continuing to culture at 37°C for 4 hours, the supernatant was discarded, 150 μL of dimethyl sulfoxide was added, shaken on a shaker at room temperature for 15 minutes, and the absorbance value A at 570 nm was measured on a microplate reader (Thermo Labsystems, Multiskan MK3). The experiment was repeated 3 times, and each experiment was provided with 3 wells of no-drug control wells and 3 wells of cell-free blank wells. According to the formula: inhibition rate%=(A _{control group} -A _{administration group} )/(A _{control group} -A _{blank group} )×100%, the drug's inhibition rate on cell proliferation was calculated and the half inhibitory concentration (IC ₅₀ ) was calculated. The results are shown in Table 1. It can be concluded from the table that MCC-LDM and lidamycin exhibit comparable cytotoxic activity, M(PEG) ₂ -LDM activity is slightly weaker, and LDM-HSA activity is somewhat enhanced.

Table 1 Cytotoxic activity of lidamycin prodrugs and preformed conjugates on tumor cells

Example 8. Distribution of lidamycin prosthetic protein prodrug in tumor-bearing mice

In this example, first, fluorescent molecules are used to label lidamycin prosthetic protein prodrug and lidamycin prosthetic protein. Take 3mg Sulfo-Cyanine7 amine (Xi'an Ruixi RH-7775) and dissolve it in 1.55mL MES buffer (0.1M MES, 0.9% NaCl, pH4.5-5), add 3.51mg MCC-rLDP or rLDP, and then add 0.15 mL of 10 mg/mL EDC (Pierce) dissolved in pure water was reacted in the dark for 2 hours at room temperature, passed through a PD-10 column (GE Healthcare), and excess unreacted fluorescein was removed to obtain MCC-rLDP-Cy7 and rLDP-Cy7 . NCI-H460 cells were inoculated subcutaneously in the axilla of BALB/c nude mice (18-22 g, Beijing Huafukang Biotechnology Co., Ltd.) at 5×10 ⁶ /only, and when the tumor grew to 200-300 mm ³ , the labeled A good protein was injected into the tail vein of the mouse, and live imaging was performed at 0.5 hours, 6 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, and 7 days respectively, and the imaging instrument IVIS-200Imaging System (Xenogen, USA) , Excitation light/emission light wavelength: 675/760nm. The results showed that MCC-rLDP-Cy7 began to accumulate at the tumor site 0.5 hours after injection, and there was a significant enrichment after 24 hours, and there was still a strong enrichment after 7 days (Figure 7); while rLDP-Cy7 was 3 days after injection No enrichment at the tumor site was found yet (Fig. 8). The above results indicated that the tumor targeting ability of MCC-rLDP was significantly stronger than that of rLDP.

Embodiment 9. Animal experimental treatment scheme of lidamycin prodrug

The in vivo efficacy of lidamycin prodrug was evaluated using human large cell lung cancer NCI-H460 xenograft tumor model. NCI-H460 cells were inoculated subcutaneously in the axilla of BALB/c nude mice (18-22 g, Beijing Huafukang Biotechnology Co., Ltd.) at 5×10 ⁶ /only. After the tumor grew to a certain size, the tumor was cut into small pieces of 2 mm ³ and inoculated into the subcutaneous area of the armpits of nude mice, a total of 18 mice. After 10 days, the tumors grew to an average of about 100mm ³ , and were divided into 3 groups according to the size of the tumors: the control group, the MCC-LDM group, and the LDM group, and were injected into the tail vein. The dosage was 0.05mg/kg, 0.2mL/piece , 1 time/week, administered twice in total. During the experiment, the long axis a and the short axis b perpendicular to it were measured every 3-4 days, and the body weight of the animals was recorded. The tumor volume and inhibition rate were calculated with the formula V=1/2ab ² ((tumor volume of the control group−tumor volume of the test group)/tumor volume of the control group×100%). The experiment was terminated 11 days after the last dose.

The experimental results show that lidamycin prodrug MCC-LDM has a significant curative effect in vivo, and can significantly inhibit the growth of NCI-H460 subcutaneous tumor at 0.05mg/kg; on the 18th day of the experiment, the tumor inhibition rate of MCC-LDM reached 64.3%. Significantly higher than Lidamycin LDM at 0.05 mg/kg (49.7%) (Figure 9). Compared with the body weight at the time of initial administration (the 0th day), the body weight of the administration group all decreased (Fig. 10); at the end of the experiment (the 18th day), the average body weight of the MCC-LDM group decreased by about 6.3%. group decreased by 9.1% (Figure 11), indicating that the systemic toxicity of MCC-LDM is weaker than that of LDM.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. a lidamycin prodrug, is characterized in that, its chemical formula is as shown in formula 1: M-L-LDM formula 1 Wherein, M represents maleimide; LDM represents lidamycin; L is a spacer arm, which is selected from carbonyl, C _1-10 carbonyl, C ₂ (PEG) _2-24 carbonyl, carbonyl substituted methyl cycloalkane Any of the groups, preferably methylcyclohexanecarboxamido, phenyl, propionamide, carbonyl-substituted methylcyclopropanyl, carbonyl-substituted methylcyclobutanyl, carbonyl-substituted methyl Cyclopentyl, carbonyl-substituted methylcyclohexyl, carbonyl-substituted methylcyclohexyl, carbonyl-substituted methylcyclooctyl, carbonyl-substituted methylcyclohexyl is more preferred. 2. lidamycin prodrug as claimed in claim 1 is characterized in that, it is made of lidamycin and the maleimide group covalently bonded thereto, and its chemical formula is as shown in formula 2: Mal-LDM Formula 2 Wherein, Mal represents a maleimide group, which includes a maleimide group and a spacer arm; LDM represents lidamycin. 3. lidamycin prodrug as described in any one of claim 1-2, it is characterized in that, described maleimide group is maleimide methylcyclohexyl, maleimide Iminomethylcyclohexanecarboxamide, maleimidephenyl, maleimidepropionamide, maleimidemethylcyclopropanyl, maleimidemethylcyclobutane radical, maleimidomethylcyclopentyl, maleimidomethylcycloheptanyl, maleimidomethylcyclooctyl; preferably, maleimidomethylcyclo Hexyl is 4-(N-maleimidomethyl)cyclohexyl. 4. lidamycin prodrug as described in any one in claim 1-3, is characterized in that, the aliphatic ring 1 position of described 4-(N-maleimidomethyl) cyclohexane group The carbonyl group is connected to the amino group on the prosthetic protein of lidamycin, and the 3-position of the five-membered heterocycle of the 4-(N-maleimidomethyl) cyclohexane group is connected to the albumin Sulfhydryl binding. 5. the preparation method of lidamycin prodrug as described in any one of claim 1-4, is characterized in that, this preparation method comprises at least one end containing the linking agent of maleimide group and lidamycin Reaction steps; Preferably, the other end of cross-linking agent contains succinimide group or sulfo (Sulfo) succinimide group; Further preferred cross-linking agent selects following compound: Succinimide 4-(N- Maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and Sulfo-SMCC, succinimide 4-(N-maleimidomethyl)cyclohexane-1-carboxylate -(6-aminocaproate) (LC-SMCC); PEGylated SMCC crosslinker SM(PEG) _2-24 ; succinimidyl 4-(p-maleimidophenyl) Butyrate (SMPB) and Sulfo-SMPB, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and Sulfo-MBS; and succinimide-6-maleimide iminopropionamidohexanoate (SMPH); and N-alpha-maleimidoacetyl-oxysuccinimide ester (AMAS), N-beta-maleimidopropionyl- Oxysuccinimide ester (BMPS), N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and Sulfo-GMBS, N-ε-maleimidocaproyl- Oxysuccinimide ester (EMCS) and Sulfo-EMCS, N-κ-maleimidoundecanoyloxy-sulfosuccinimide ester sodium salt (Sulfo-KMUS). 6. the method for preparing of lidamycin prodrug as claimed in claim 5 is characterized in that, between the maleimide group of described cross-linking agent and the succinimide ester group, be spacer arm, it selects Any of carbonyl, C _1-10 carbonyl, C ₂ (PEG) _2-24 carbonyl, carbonyl substituted methylcycloalkyl, preferably methylcyclohexanecarboxamido, phenyl, propionamide Carbonyl-substituted methylcyclopropanyl, carbonyl-substituted methylcyclobutanyl, carbonyl-substituted methylcyclopentyl, carbonyl-substituted methylcyclohexyl, carbonyl-substituted methylcycloheptanyl , carbonyl-substituted methylcyclooctyl, more preferably carbonyl-substituted methylcyclohexyl. 7. the method for the preparation of lidamycin prodrug as described in any one of claim 5-6, it is characterized in that, described linking agent is Sulfo-SMCC, preferably: get appropriate LDM and be dissolved in PBS-ETDA Solution (KH ₂ PO ₄ : 0.2g/L, Na ₂ HPO ₄ ·12H ₂ O: 2.9g/L, NaCl: 8.0g/L, KCl: 0.2g/L, 0.58g/L EDTA, pH 7.2), add Sulfo-SMCC dissolved in pure water, wherein the molar ratio of LDM to Sulfo-SMCC is 1:0.1~1000, preferably 1:1~100, react in the dark at 4~37°C for 1 minute to 48 hours, preferably The lidamycin prodrug can be prepared by reacting at room temperature in the dark for 10 to 120 minutes. 8. The preparation method of lidamycin prodrug according to any one of claims 5-7, characterized in that the preparation method further comprises a separation and purification step of removing small molecules by gel filtration chromatography or ultrafiltration. 9. Use of the lidamycin prodrug according to any one of claims 1-4 in the preparation of antitumor drugs. 10. The application of the preformed conjugate of lidamycin prodrug and albumin as described in any one of claims 1-4.

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