A tetrasulfide-bridged dimer prodrug, self-assembled nanoparticles thereof and applications thereof

Abstract

The application discloses a kind of four sulfur bond bridging dimer prodrugs and its self-assembly nanoparticles and application, belong to medical technology field.The four sulfur bond bridging dimer prodrug and its self-assembly nanoparticles are designed and synthesized, the ideal four sulfur bond is obtained by screening four sulfur bond chemical stability, and it is applied to the construction of prodrug self-assembly nanoparticles, and then the various applications in drug delivery are realized.The influence of different kinds of four sulfur bond and four sulfur bond compared with three sulfur bond and disulfide bond on the stability of prodrug self-assembly nanoparticles, drug release, pharmacokinetics and tissue distribution, and the influence of the ultimate antitumor effect in vivo and in vitro is explored, and the mechanism and effect of four sulfur bond bridging prodrug self-assembly nanoparticles drug combined with reducing substance are explored, to provide a variety of choices for developing high-efficiency low-toxicity prodrug self-assembly nanoparticles, promote the application of prodrug self-assembly nanoparticles in tumor treatment, meet the urgent needs of high-end cancer chemotherapy preparations in clinic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a kind of four-sulfur bond bridged dimer prodrug and its self-assembly nanoparticles and application, specifically relates to a kind of four-sulfur bond bridged reduced hypersensitive cabazitaxel dimer prodrug and the construction of dimer prodrug self-assembly nanoparticles, and its single or with the application in drug delivery of reducing material. BACKGROUND

[0002] Chemotherapy plays a vital role in clinical treatment, especially for advanced malignant tumors, and as an adjuvant therapy after radical surgery. However, chemotherapy is limited by serious side effects, causing great pain and suffering to patients. In addition, the poor physicochemical properties of chemotherapy drugs also limit their delivery efficiency in the body. For example, the second-generation taxane drug cabazitaxel (CTX) shows stronger anticancer activity compared with paclitaxel and docetaxel, and is used for second-line treatment of prostate cancer, with great market potential. However, in addition to the desired therapeutic effect, cabazitaxel can also cause some toxic side effects, including anemia, leukopenia, neutropenia, thrombocytopenia, and diarrhea. At the same time, the poor physicochemical properties of cabazitaxel also limit their delivery in the body. In order to solve the problem of poor solubility of cabazitaxel, the cabazitaxel injection (Jevtana®) used in clinic contains a large amount of Tween 80 (60 mg / 1.56 g: CTX / Tween 80), which is easy to cause serious allergic reactions. Therefore, it is urgent to develop an efficient and low-toxic drug delivery system.

[0003] In recent years, the application of prodrug strategy and nanotechnology in drug delivery has attracted great attention. Prodrug strategy can effectively improve the adverse properties of drugs, but prodrug molecules are easily eliminated in vivo and have low tumor targeting efficiency. Nanodrug delivery system (NDDS) based on nanotechnology can prolong the in vivo circulation time of drugs and improve the accumulation of drugs at tumor sites through active or passive targeting. However, traditional nanomedicines loaded with drugs by physical embedding have low drug loading capacity (usually less than 10%) and safety concerns of nanomaterials used in vivo. Prodrug self-assembly nanotechnology combines the advantages of prodrug strategy and nanotechnology, which modifies drug molecules into prodrug molecules with self-assembly ability, and forms nanoparticles through self-assembly process, showing significant effects in simplifying the preparation process, improving drug loading capacity and reducing the toxicity of excipients. Dimer prodrug connects two drug molecules together. On the one hand, compared with monomer prodrug nanoparticles, the proportion of drugs in dimer prodrug self-assembly nanoparticles increases significantly, so it has absolute advantages in improving drug loading capacity and enhancing efficacy. On the other hand, due to the influence of the physicochemical properties of drugs, the self-assembly ability of dimer prodrug is poor, and it is not easy to form stable prodrug self-assembly nanoparticles, which makes the construction of dimer prodrug self-assembly nanoparticles more dependent on chemical bridging to improve the self-assembly ability of prodrug. In addition, prodrugs have no or low biological activity and need to be metabolized into active substances in vivo to exert pharmacological effects. In order to achieve high-efficiency antitumor effect and reduce toxic side effects, prodrug self-assembly nanoparticles must remain stable in vivo, and the active parent drug must be released selectively by chemical bridging in response to tumor-specific signals. Therefore, the key to constructing ideal dimer prodrug self-assembly nanoparticles is to use efficient chemical bridging to improve the self-assembly stability of dimer prodrug and make prodrug self-assembly nanoparticles be able to selectively activate and release the parent drug at tumor sites.

[0004] Sulfur bond has a bond angle and dihedral angle of nearly 90°, and the introduction of sulfur bond in drug molecules can construct effective spatial conformation, balance intermolecular forces, and promote prodrug self-assembly. In addition, high levels of glutathione (GSH) is an important feature that distinguishes tumor cells from normal cells, and sulfur bond is chemically sensitive to high levels of GSH, which can achieve intelligent response and selective activation of tumors. Therefore, the introduction of sulfur bond in the prodrug self-assembly nanoparticle system is an effective means to construct ideal prodrug self-assembly nanoparticles. However, the widely used single sulfur bond and disulfide bond have very limited ability to improve the self-assembly ability of dimeric prodrugs, the key problem of poor self-assembly ability of dimeric prodrugs has not been solved, and the response sensitivity of single sulfur bond and disulfide bond to tumor environment also needs to be improved. Therefore, it is urgent to develop efficient new sulfur bonds to overcome the challenges faced by prodrug self-assembly nanomedicine delivery systems and improve drug efficiency. The trisulfide bond, which adds one sulfur atom to the disulfide bond, can improve the assembly ability and reduction sensitivity of the prodrug. Therefore, whether the tetrasulfide bond, which adds one sulfur atom to the trisulfide bond, can achieve better prodrug delivery effect? However, there is no related report on intelligent response prodrug and self-assembly nanomedicine delivery system based on tetrasulfide bond. Reduced substances such as ascorbic acid (AsA) are reported to promote tumor cell apoptosis and synergize with chemotherapy, and other reduced substances such as GSH and cysteine (Cys) are also used in clinical applications. Therefore, if the reduced-sensitive prodrug self-assembly nanoparticles are combined with reduced substances, can it further promote the release of active drugs at the tumor site and achieve better therapeutic effect, and produce multi-mechanism synergistic effect? These scientific problems are worthy of further research. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a tetrasulfide-bridged dimeric prodrug, a self-assembly nanoparticle thereof and application, which solves the technical problems of poor self-assembly ability, poor response sensitivity to tumor environment and low drug delivery efficiency of dimeric prodrugs with single sulfur bond and disulfide bond.

[0006] To achieve the above object, the technical scheme adopted by the present application comprises:

[0007] In a first aspect, the present application provides a tetrasulfide-bridged dimeric prodrug, the structure general formula of which is shown as general formula I:

[0008]

[0009] General formula I

[0010] In the formula, the tetrasulfide bond is , or ;

[0011] In the formula, the drug is an antitumor drug, an antimetabolite drug or an anti-inflammatory drug containing a hydroxyl group, an amino group or a carboxyl group, the antitumor drug is a taxane, an anthraquinone, a nucleoside, a camptothecin, a platinum, a vinca alkaloid, a podophyllotoxin or an artemisinin compound; the antimetabolite drug is a pyrimidine, a purine, a thymidine or a folic acid; the anti-inflammatory drug is halofantrine or a derivative thereof, griseofulvin or a derivative thereof, or distamycin A or a derivative thereof.

[0012] Further, the drug is a taxane or an anthraquinone compound, the taxane compound includes paclitaxel, docetaxel, cabazitaxel, larotaxel, sinapultimil, conumaclast or milataxel; the anthraquinone compound includes daunorubicin, doxorubicin, epirubicin, pyran doxorubicin or aclarubicin.

[0013] Specifically, the drug is cabazitaxel, and the structural formula is as follows:

[0014] 、 or .

[0015] In a second aspect, the present application provides a preparation method of the four-sulfur bond bridged dimer prodrug, comprising the following steps:

[0016] The binary acid containing a four-sulfur bond is dissolved in a solvent and stirred uniformly; 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and the drug are dissolved in a solvent and stirred uniformly, and then mixed with the binary acid solution containing a four-sulfur bond, stirred at room temperature for 10-12 h under N2 protection; 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride are added to the mixed solution, and stirring is continued at room temperature for 12-24 h under N2 protection, and then separated and purified to obtain the product.

[0017] Specifically, the present application provides a synthesis method of the binary acid containing a four-sulfur bond:

[0018] Sodium thiosulfate pentahydrate and 3-bromopropionic acid / 4-bromobutyric acid / 5-bromovaleric acid are respectively dissolved in pure water, and the 3-bromopropionic acid / 4-bromobutyric acid / 5-bromovaleric acid solution is slowly added dropwise to the sodium thiosulfate pentahydrate solution under stirring at an oil bath of 50-60℃, and the reaction is carried out for 4-8 h under N2 protection. After the reaction is completed, the above reaction solution is cooled to 25℃, sodium sulfide nonahydrate (0.3 mmol) is dissolved in 10 mL of pure water, and then added dropwise to the above cooled reaction solution, and the reaction is carried out for 12 h at 25℃ overnight. After the reaction is completed, ethyl acetate is used for extraction, and the ethyl acetate layers are combined and rotary dried to obtain the crude product of the four-sulfur binary acid. The four-sulfur binary acid is separated and purified by preparative liquid chromatography.

[0019] Further, the binary acid containing a tetrasulfide bond is 3,3'-tetrasulfide dipropionic acid, 4,4'-tetrasulfide dibutyric acid or 5,5'-tetrasulfide dipentanoic acid.

[0020] In a third aspect, the present application provides a tetrasulfide bond bridged dimer prodrug self-assembled nanoparticle, including a PEG modified small molecule prodrug nanoparticle, an actively targeted small molecule prodrug nanoparticle, a small molecule prodrug nanoparticle loaded with a fluorescent substance or a hydrophobic drug;

[0021] The PEG modified small molecule prodrug nanoparticle is a uniform nanoparticle spontaneously formed by dropping a mixed solution of the tetrasulfide bond bridged dimer prodrug and a PEG modifier into water; the PEG modifier is TPGS, DSPE-PEG, PLGA-PEG or PE-PEG, and the preferred PEG modifier is DSPE-PEG. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000 and 5000, and more preferably the molecular weight of the PEG is 2000.

[0022] The actively targeted small molecule prodrug nanoparticle is a uniform nanoparticle spontaneously formed by dropping a mixed solution of the tetrasulfide bond bridged dimer prodrug and a PEG with a target head and a PEG modifier into water; the PEG modifier is TPGS, DSPE-PEG, PLGA-PEG or PE-PEG, and the PEG modifier with a target head includes but is not limited to DSPE-PEG-FA, DSPE-PEG-Biotin, cRGD-PEG-PLL, DSP-PEG 2k -Pep-1 or DSPE-PEG-CREKA.

[0023] The molecular weight of the PEG is 1000-5000; and the weight ratio of the tetrasulfide bond bridged dimer prodrug to the PEG modifier is 90:10-70:30. Under this range, the prodrug self-assembled nanoparticle can exert a better antitumor effect.

[0024] The small molecule prodrug nanoparticle loaded with a fluorescent substance or a hydrophobic drug is obtained by self-assembly after blending the tetrasulfide bond bridged dimer prodrug with a fluorescent substance or a hydrophobic drug; the fluorescent substance is coumarin-6, rhodamine, DiR, DiI, Cy-5 or Cy-7, and the hydrophobic drug is camptothecin, hydroxycamptothecin or doxorubicin.

[0025] More specifically, the preparation method of the PEG modified tetrasulfide bond bridged dimer prodrug self-assembled nanoparticle is as follows:

[0026] The four-sulfur bond bridged dimer prodrug and the PEG modifying agent are dissolved in a solvent, and the solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles, and the solvent is removed by distillation under reduced pressure to obtain a nanocolloid solution without organic solvent.

[0027] The solvent is one or more of ethanol, dimethyl sulfoxide, N,N'-dimethylformamide, tetrahydrofuran or acetone.

[0028] More specifically, the preparation method of the small molecule prodrug nanoparticle loaded with a fluorescent substance or a hydrophobic drug is as follows: a certain amount of fluorescent substance or hydrophobic drug is dissolved in a proper amount of ethanol, and the ethanol solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles. The ethanol in the preparation is removed by dialysis to obtain a nanocolloid solution without any organic solvent.

[0029] In a fourth aspect, the present application provides a pharmaceutical composition comprising the four-sulfur bond bridged dimer prodrug and a pharmaceutically acceptable carrier or excipient.

[0030] In a fifth aspect, the present application provides a use of the four-sulfur bond bridged dimer prodrug or the dimer prodrug self-assembled nanoparticle or the pharmaceutical composition in the preparation of a drug delivery system.

[0031] In a sixth aspect, the present application provides a use of the four-sulfur bond bridged dimer prodrug or the dimer prodrug self-assembled nanoparticle or the pharmaceutical composition in the preparation of an antitumor drug.

[0032] In a seventh aspect, the present application provides a use of the four-sulfur bond bridged dimer prodrug or the dimer prodrug self-assembled nanoparticle or the pharmaceutical composition in the preparation of an injection, oral administration or topical administration system.

[0033] In an eighth aspect, the present application provides a use of the four-sulfur bond bridged dimer prodrug or the dimer prodrug self-assembled nanoparticle or the pharmaceutical composition in the treatment of a tumor drug in combination with a reducing agent, wherein the reducing agent comprises at least one of glutathione, cysteine, ascorbic acid, vitamin E, dithiothreitol, carotenoid or thioctic acid.

[0034] The present application has the following beneficial effects:

[0035] (1) The present application obtains ideal four-sulfur bonds by designing and synthesizing four-sulfur bonds and screening the chemical stability of the four-sulfur bonds, and applies the four-sulfur bonds to the construction of prodrug self-assembled nanoparticles, and then realizes the application in drug delivery. The present application explores the influence of different types of four-sulfur bonds, three-sulfur bonds and disulfide bonds on the stability, drug release, pharmacokinetics and tissue distribution of prodrug self-assembled nanoparticles, and then determines the antitumor effect in vitro and in vivo, and explores the mechanism and effect of the combination of four-sulfur bond bridged prodrug self-assembled nanoparticles and exogenous reducing agent, so as to provide multiple choices for developing high-efficiency and low-toxicity prodrug self-assembled nanoparticles, promote the application of prodrug self-assembled nanoparticles in tumor treatment, and provide a theoretical basis for various treatment modes of prodrug self-assembled nanoparticles.

[0036] (2) The dimeric prodrug containing different types of four-sulfur bond bridges is designed and synthesized, and the synthesis method is simple and easy to implement.

[0037] (3) Based on γ-4S-2CTX, the dimeric prodrug γ-3S-2CTX and γ-2S-2CTX bridged by three-sulfur bond and disulfide bond are designed and synthesized as controls, which illustrates the advantage of introducing four-sulfur bond into the structure of prodrug to construct prodrug self-assembled nanoparticles.

[0038] (4) The uniform dimeric prodrug self-assembled nanoparticles are prepared, the preparation method is simple and easy to implement, the efficient drug loading is realized, the ultra-high drug loading capacity is more than 70%, the stability in 10% FBS phosphate buffer solution is 24 h, and the particle size basically remains unchanged after being placed at room temperature for 4 weeks.

[0039] (5) The differences of different chemical bridges in stability, self-assembly, redox sensitive response ability and antitumor activity, and the influence on the stability, drug release, cytotoxicity, pharmacokinetics, tissue distribution and pharmacodynamics of prodrug self-assembled nanoparticles are investigated.

[0040] (6) Based on the ultra-sensitive redox response ability of four-sulfur bond prodrug, the influence of four-sulfur bond on pharmacodynamics in vitro and in vivo combined with reducing agent is investigated. According to the experimental results, γ-4S-2CTX has the best assembly ability, good blood circulation stability and ultra-sensitive redox response ability. At the same time, the nanoparticles of γ-4S-2CTX combined with reducing agent have a significant improvement in drug efficacy. The present application provides multiple choices for developing high-efficiency and low-toxicity prodrug self-assembled nanoparticles, promotes the application of prodrug self-assembled nanoparticles in tumor treatment, and provides a theoretical basis for various treatment modes of prodrug self-assembled nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The structure of 3,3'-four-sulfur bond bridged cabazitaxel dimeric prodrug (β-4S-2CTX) in Example 1 of the present application is confirmed; wherein A is the structure of β-4S-2CTX1 H-NMR spectrum; B is the mass spectrum of β-4S-2CTX; C is the high performance liquid chromatography purity chart of β-4S-2CTX; D is the sulfur exchange impurity during the placement of β-4S-2CTX.

[0042] Figure 2 For structural confirmation of 4,4'-tetrasulfide bridged cabazitaxel dimer prodrug (γ-4S-2CTX) in Example 2 of the application; wherein A is the H-NMR spectrum of γ-4S-2CTX; B is the mass spectrum of γ-4S-2CTX; C is the high performance liquid chromatography purity chart of γ-4S-2CTX. 1 H-NMR spectrum; B is the mass spectrum of γ-4S-2CTX; C is the high performance liquid chromatography purity chart of γ-4S-2CTX.

[0043] Figure 3 For structural confirmation of 5,5'-tetrasulfide bridged cabazitaxel dimer prodrug (δ-4S-2CTX) in Example 3 of the application; wherein A is the H-NMR spectrum of δ-4S-2CTX; B is the mass spectrum of δ-4S-2CTX; C is the high performance liquid chromatography purity chart of δ-4S-2CTX. 1 H-NMR spectrum; B is the mass spectrum of δ-4S-2CTX; C is the high performance liquid chromatography purity chart of δ-4S-2CTX.

[0044] Figure 4 For structural confirmation of 4,4'-trisulfide bridged cabazitaxel dimer prodrug (γ-3S-2CTX) in Example 4 of the application; wherein A is the H-NMR spectrum of γ-3S-2CTX; B is the mass spectrum of γ-3S-2CTX; C is the high performance liquid chromatography purity chart of γ-3S-2CTX. 1 H-NMR spectrum; B is the mass spectrum of γ-3S-2CTX; C is the high performance liquid chromatography purity chart of γ-3S-2CTX.

[0045] Figure 5 For structural confirmation of 4,4'-disulfide bridged cabazitaxel dimer prodrug (γ-2S-2CTX) in Example 4 of the application; wherein A is the H-NMR spectrum of γ-2S-2CTX; B is the mass spectrum of γ-2S-2CTX; C is the high performance liquid chromatography purity chart of γ-2S-2CTX. 1 H-NMR spectrum; B is the mass spectrum of γ-2S-2CTX; C is the high performance liquid chromatography purity chart of γ-2S-2CTX.

[0046] Figure 6 Transmission electron microscope chart of self-assembled nanometer of tetrasulfide bridged cabazitaxel dimer prodrug prepared in Example 7 of the application.

[0047] Figure 7 Particle size-colloidal stability chart of PEG modified tetrasulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the application; wherein a is the room temperature storage stability of tetrasulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles; b is the rat plasma stability of tetrasulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles.

[0048] Figure 8Figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; wherein, a is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in blank release medium; b is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 10 mM H2O2 condition; c is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 1 mM GSH condition; d is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 500 μM GSH condition; e is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 1 mM Cys condition; f is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 500 μM Cys condition; g is the figure for in vitro release test of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under 20 mM AsA condition.

[0049] Figure 9 Figure for blood concentration-time curve of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present application; wherein, a is the figure for blood concentration-time curve of the prodrug of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; b is the figure for blood concentration-time curve of cabazitaxel of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; c is the figure for blood concentration-time curve of total amount of the prodrug and cabazitaxel of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles.

[0050] Figure 10 Figure for biodistribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present application; wherein, a is the heart distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; b is the liver distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; c is the spleen distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; d is the lung distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; e is the kidney distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; f is the tumor distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; g is the heart distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles loaded with DiR; h is the liver distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles loaded with DiR; i is the spleen distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles loaded with DiR; j is the lung distribution of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles loaded with DiR.

[0051] Figure 11 Figure for 4T1 tumor cell uptake of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present application.

[0052] Figure 12 Figure 7 is a cytotoxicity diagram of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present application; wherein a is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles on tumor cells 4T1 cells, RM-1 cells, KB cells and Panc02 cells; b is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles on normal cells L02 cells; c is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on RM-1 cells; d is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with AsA on RM-1 cells; e is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on 4T1-1 cells; f is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with GSH on 4T1 cells; g is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on L02 cells; h is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with GSH on L02 cells; i is the half maximal inhibitory concentration of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with AsA on L02 cells.

[0053] Figure 13 Figure 8 is the anti-tumor effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 of the present application in the RM-1 mouse model; wherein a is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles on the growth of RM-1 mouse prostate cancer subcutaneous tumors; b is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles on the tumor-bearing rate of RM-1 mice; c is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles on the body weight of RM-1 tumor-bearing mice; d is the tumor photo of Balb / C tumor-bearing mice after treatment with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles; e is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on the growth of RM-1 mouse prostate cancer subcutaneous tumors; f is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on the tumor-bearing rate of RM-1 mice; g is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys on the body weight of RM-1 tumor-bearing mice; h is the tumor photo of Balb / C tumor-bearing mice after treatment with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with Cys.

[0054] Figure 14Anti-tumor effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in Example 7 on a Balb / C mouse model; wherein, a is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) on the growth of breast cancer subcutaneous tumors of Balb / C mice; b is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) on the body weight of tumor-bearing mice; c is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) on the tumor-bearing rate of Balb / C mice; d is the tumor photo of Balb / C tumor-bearing mice after treatment with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose); e is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (high dose) on the growth of breast cancer subcutaneous tumors of Balb / C mice; f is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (high dose) on the body weight of tumor-bearing mice; g is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (high dose) on the tumor-bearing rate of Balb / C mice; h is the tumor photo of Balb / C tumor-bearing mice after treatment with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (high dose); i is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) combined with AsA on the growth of breast cancer subcutaneous tumors of Balb / C mice; j is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) combined with AsA on the body weight of tumor-bearing mice; k is the effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) combined with AsA on the tumor-bearing rate of Balb / C mice; l is the tumor photo of Balb / C tumor-bearing mice after treatment with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles (low dose) combined with AsA. DETAILED DESCRIPTION

[0055] In order to better explain the present application, so as to be understood, the present application will be described in detail below by specific embodiments in combination with the drawings.

[0056] In order to better understand the above technical solutions, the exemplary embodiments of the present application will be described in more detail below. It should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application can be more clearly, thoroughly understood, and the scope of the present application can be completely conveyed to those skilled in the art.

[0057] Example 1: Synthesis of 3,3'-four-sulfur bond bridged cabazitaxel dimer prodrug (β-4S-2CTX)

[0058] 0.25 mmol of 3,3'-tetrathiodipropionic acid was dissolved in 10 mL of dichloromethane, 0.05 mmol of 4-dimethylaminopyridine (DMAP) and 1 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 0.5 mmol of cabazitaxel were dissolved in 20 mL of anhydrous dichloromethane, and mixed with the dichloromethane solution of 3,3'-tetrathiodipropionic acid, and stirred at room temperature for 10-12 h. Then 0.5 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.05 mmol of 4-dimethylaminopyridine were added, and stirred at room temperature for 12-24 h. The above reaction was carried out under N2 protection, and the obtained product (named β-4S-2CTX) was separated and purified by preparative liquid chromatography, and the structural formula is shown below.

[0059]

[0060] The structure of β-4S-2CTX prepared in Example 1 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrum, and the results are shown in Figure 1

[0061] The nuclear magnetic resonance spectrum analysis results are as follows: 1 ​H NMR (400 MHz, CDCl3): δ 8.118 and 8.099(d, 2H, Ar-H), 7.609 (t, 1H, Ar-H), 7.500 (t, 2H, Ar-H), J = 7.6 Hz, 7.405(t, 2H, Ar-H, J = 7.6 Hz), 7.307 (s, 3H, Ar-H), 6.258 (t, 1H, H-13), 5.654and 5.637 (d, 1H, H-2, J = 6.8 Hz), 5.373 (s, 1H, -NH-C(O)-), 5.009 and 4.986(d, 1H, H-5), 4.819 (s, 1H, H-10), 4.323 and 4.302 (d, 1H, H-21), 4.181 and4.160 (d, 1H, H-20b, J = 8.4 Hz), 3.901 (m, 1H, H-20a, J = 8.4 Hz), 3.853 and3.836 (d, 1H, H-3, J = 7.6 Hz), 3.434 (s, 3H, H-28), 3.298 (s, 3H, H-27),3.096 (t, 2H, H-6), 2.706 (m, 3H, H-21), 2.441 (s, 2H, α-CH2), 1.993 (s, 2H,β-CH2), 2.009 and 1.995 (d, 3H, H-18), 1.792 (t, 1H, 1-OH), 1.713 (s, 3H, H-19), 1.352 (s, 9H, H-4’), 1.210 and 1.197 (d, 6H, H-16 and H-17).

[0062] Mass spectrum results MS (ESI) m / z [M+Na] + =1919.59037. Characteristic peaks of β-3S-2CTX and β-5S-2CTX were also found in the MS scan and high performance liquid chromatography analysis, indicating that the chemical structure of β-4S-2CTX was partially transformed. MS (ESI) β-3S-2CTX (C 96 H 120 N2O 30 S3) MS (ESI) m / z [M+K] + = 1916, β -5s-2CTX(C 96 H 120 N2O30 S5) MS (ESI) m / z [M+Na] + = 1964. Therefore, it was excluded in the subsequent experiments.

[0063] Example 2: Synthesis of 4,4'-tetrasulfide bridge linked cabazitaxel dimer prodrug (γ-4S-2CTX)

[0064] Using the preparation method of Example 1, 3,3'-tetrasulfide dipropionic acid was replaced by 4,4'-tetrasulfide dibutyric acid to prepare 4,4'-tetrasulfide bridge linked cabazitaxel dimer prodrug, named γ-4S-2CTX, the structural formula is shown as follows.

[0065]

[0066] Mass spectrometry and nuclear magnetic resonance hydrogen spectrum were used to determine the structure of γ-4S-2CTX prepared in Example 2, and the results are shown in Figure 2 .

[0067] The nuclear magnetic resonance spectrum analysis results are as follows: 1H NMR (400 MHz, CDCl3): δ 8.118 and 8.099(d, 2H, Ar-H), 8.003 (t, 1H, Ar-H), 7.610 (t, 2H, Ar-H), J = 7.6 Hz, 7.509(t, 2H, Ar-H, J = 7.6 Hz), 7.405 (t, 2H, Ar-H), 7.308 (s, 1H, Ar-H), 6.259(s, 1H, H-13), 5.654 and 5.638 (d, 1H, H-2, J = 6.8 Hz), 5.484 (d, 1H, -NH-C(O)-), 5.361 (s, 1H, H-3’, J = 7.2 Hz), 5.016 and 4.993 (d, 1H, H-5), 4.827(s, 1H, H-10), 4.326 and 4.305 (d, 1H, H-21), 4.185 and 4.164 (d, 1H, H-20b,J = 8.4 Hz), 3.893 (m, 1H, H-20a, J = 8.4 Hz), 3.877 and 3.875 (d, 1H, H-3, J= 7.6 Hz), 3.436 (s, 3H, H-28), 3.300 (s, 3H, H-27), 2.883 (m, 2H, H-6),2.710 (m, 2H, α-CH2), 2.572 (m, 2H, γ-CH2), 2.446 (s, 3H, H-21), 2.067 (t,2H, β-CH2), 2.009 and 1.995 (d, 3H, H-18), 1.792 (t, 1H, 1-OH), 1.713 (s, 3H,H-19), 1.349 (s, 9H, H-4’), 1.211 and 1.198 (d, 6H, H-16 and H-17).

[0068] Mass spectrum results: MS (ESI) m / z [M+H] + = 1937.72574. The purity results showed that the purity of γ-4S-2CTX was 98.100%, which met the requirements of subsequent experiments.

[0069] Example 3: Synthesis of 5,5’-tetrasulfide bridge linked cabazitaxel dimer prodrug (δ-4S-2CTX)

[0070] The preparation method of Example 1 was adopted, 3, 3'-tetrathiodipropionic acid was replaced by 5, 5'-tetrathiodivaleric acid to prepare 5, 5'-tetrathio bridge connected cabazitaxel dimer prodrug, named δ-4S-2CTX, the structural formula is shown as follows.

[0071]

[0072] Mass spectrometry and nuclear magnetic resonance hydrogen spectrum method were used to determine the structure of δ-4S-2CTX prepared in Example 3, and the results are shown in Figure 3

[0073] The nuclear magnetic resonance spectrum analysis results are as follows: 1 ​H NMR (400 MHz, CDCl3): δ 8.119 and 8.101(d, 2H, Ar-H), 7.606 (t, 1H, Ar-H), J = 7.6 Hz, 7.497 (t, 2H, Ar-H, J = 7.6Hz), 7.402 (t, 2H, Ar-H), 7.304 (s, 2H, Ar-H), 7.261 (s, 1H, Ar-H), 6.264 (t,1H, H-13), 5.655 and 5.638 (d, 1H, H-2, J = 6.8 Hz), 5.475 (s, 1H, -NH-C(O)-), 5.300 (s, 1H, H-3’, J = 7.2 Hz), 5.010 and 4.998 (d, 1H, H-5), 4.824(s, 1H, H-10), 4.323 and 4.301 (d, 1H, H-21), 4.183 and 4.161 (d, 1H, H-20b,J = 8.4 Hz), 3.907 (m, 1H, H-20a, J = 8.4 Hz), 3.858 and 3.841 (d, 1H, H-3, J= 7.6 Hz), 3.437 (s, 3H, H-28), 3.301 (s, 3H, H-27), 2.880 (t, 2H, H-6),2.707 (m, 2H, α-CH2), 2. 444 (s, 2H, δ-CH2), 2.392 (s, 2H, β-CH2), 2.000 (s,2H, γ-CH2), 1.791 (t, 1H, 1-OH), 1.714 (s, 6H, H-19), 1.348 (s, 9H, H-4’),1.214 and 1.211 (d, 6H, H-16 and H-17).

[0074] Mass spectrometry results were MS (ESI) m / z [M+H] + = 1965.75599. The purity results showed that the purity of δ-4S-2CTX was 99.135%, which met the requirements of subsequent experiments.

[0075] Example 4: Synthesis of 4,4’-trisulfide bridge linked cabazitaxel dimer prodrug (γ-3S-2CTX)

[0076] Using the preparation method of Example 1, 3, 3'-tetra-thiobispropionic acid was replaced by 4, 4'-tri-thiobisbutyric acid to prepare 4, 4'-tri-thio-bridged cabazitaxel dimer prodrug, named γ-3S-2CTX, the structural formula is shown as follows.

[0077]

[0078] Mass spectrometry and nuclear magnetic resonance hydrogen spectrum were used to determine the structure of γ-3S-2CTX prepared in Example 4, and the results are shown in Figure 4 .

[0079] The nuclear magnetic resonance spectrum analysis results are as follows: 1H NMR (400 MHz, CDCl3): δ 8.119 and 8.100(d, 2H, Ar-H), 7.607 (t, 1H, Ar-H), 7.498 (t, 2H, Ar-H), J = 7.6 Hz, 7.402(t, 2H, Ar-H, J = 7.6 Hz), 7.306 (t, 2H, Ar-H), 7.263 (s, 1H, Ar-H), 6.262(t, 2H, H-13), 5.655 and 5.638 (d, 1H, H-2, J = 6.8 Hz), 5.481 (s, 1H, -NH-C(O)-), 5.360 (s, 1H, H-3’, J = 7.2 Hz), 5.012 and 4.989 (d, 1H, H-5), 4.823(s, 1H, H-10), 4.324 and 4.303 (d, 1H, H-21), 4.183 and 4.162 (d, 1H, H-20b,J = 8.4 Hz), 3.905 (m, 1H, H-20a, J = 8.4 Hz), 3.856 and 3.837 (d, 1H, H-3, J= 7.6 Hz), 3.436 (s, 3H, H-28), 3.300 (s, 3H, H-27), 2.811 (m, 2H, H-6),2.708 (m, 2H, α-CH2), 2.562 (m, 2H, γ-CH2), 2.446 (s, 3H, H-21), 2.008 (s,2H, β-CH2), 1.999 (t, 3H, H-18), 1.791 (t, 2H, 1-OH), 1.714 (s, 3H, H-19),1.348 (s, 9H, H-4’), 1.213 and 1.199 (d, 6H, H-16 and H-17).

[0080] Mass spectrometry results were MS (ESI) m / z [M+H] + = 1905.75255. The purity results showed that the purity of γ-3S-2CTX was 98.703%, which met the requirements of subsequent experiments.

[0081] Example 5: Synthesis of 4,4’-disulfide bridge linked cabazitaxel dimer prodrug (γ-2S-2CTX)

[0082] Using the preparation method of Example 1, 3, 3'-tetra-thio-dipropionic acid was replaced by 4, 4'-dithio-dibutyric acid to prepare 4, 4'-disulfide bridge linked cabazitaxel dimer prodrug, named γ-2S-2CTX, the structural formula is shown as follows.

[0083]

[0084] Mass spectrometry and nuclear magnetic resonance hydrogen spectrum were used to determine the structure of γ-2S-2CTX prepared in Example 5, and the results are shown in Figure 5

[0085] The nuclear magnetic resonance spectrum analysis results are as follows: 1 ​H NMR (400 MHz, CDCl3): δ 8.121 and 8.102(d,2H, Ar-H), 7.608 (t, 1H, Ar-H), 7.500 (t, 2H, Ar-H), J = 7.6 Hz, 7.403 (t,2H, Ar-H, J = 7.6 Hz), 7.308 (t, 2H, Ar-H), 7.263 (s, 1H, Ar-H), 6.263 (s,2H, H-13), 5.655 and 5.638 (d, 1H, H-2, J = 6.8 Hz), 5.480 (s, 1H, -NH-C(O)-), 5.353 (s, 1H, H-3’, J = 7.2 Hz), 5.015 and 4.992 (d, 1H, H-5), 4.822(s, 1H, H-10),4.324 and 4.303 (d, 1H, H-21), 4.183 and 4.162 (d, 1H, H-20b, J= 8.4 Hz), 3.903 (m, 1H, H-20a, J = 8.4 Hz), 3.854 and 3.837 (d, 1H, H-3, J =7.6 Hz), 3.435 (s, 3H, H-28), 3.299 (s, 3H, H-27), 2.706 (m, 2H, α-CH2),2.582 (m, 2H, H-6, J = 8.4 Hz), 2.534 (m, 2H, γ-CH2), 2.441 (s, 3H, H-21),2.045 (m, 2H, β-CH2), 1.993 (t, 3H, H-18), 1.790 (t, 1H, 1-OH), 1.712 (s, 3H,H-19), 1.344 (s, 9H, H-4’), 1.211 and 1.197 (d, 6H, H-16 and H-17).

[0086] Mass spectrometry results were MS (ESI) m / z [M+H] + = 1873.78419. The purity results showed that the purity of γ-3S-2CTX was 99.431%, which met the requirements of subsequent experiments.

[0087] Example 6: Preparation of non-PEG modified / PEG modified four-thio-linkage bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0088] (1) Preparation of non-PEG modified tetra-sulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0089] The γ-4S-2CTX, δ-4S-2CTX, γ-3S-2CTX and γ-2S-2CTX were dissolved and diluted to 10, 5 and 2.5 mg / mL (volume: 0.8 mL), respectively. Then the prodrug solution was slowly added to 3.2 mL deionized water (final concentration: 2, 1 and 0.5 mg / mL) under vigorous stirring (1000 rpm). As shown in Table 1, γ-4S-2CTX and δ-4S-2CTX have the strongest self-assembly ability, and can form uniform nanoparticles at these concentrations, followed by γ-3S-2CTX, which can form nanoparticles at a concentration of 0.5 mg / mL, but aggregates and precipitates at a larger concentration. And γ-2S-2CTX cannot self-assemble at these three concentrations, indicating that the disulfide bridged cabazitaxel dimer prodrug has poor assembly ability.

[0090] Table 1 Particle size and particle size distribution of non-PEG modified dimer prodrug self-assembled nanoparticles

[0091]

[0092] (2) Preparation of PEG modified tetra-sulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0093] Precisely weigh DSPE-PEG 2k 1 mg and tetra-sulfide bridged cabazitaxel dimer prodrug 4 mg were dissolved and mixed with 1 mL ethanol, and the ethanol solution was slowly added to 4 mL deionized water under stirring, i.e. uniform nanoparticles γ-4S-2CTX nanoparticles, δ-4S-2CTX nanoparticles, γ-3S-2CTX nanoparticles and γ-2S-2CTX nanoparticles were formed spontaneously. The ethanol in the nanoparticle preparation was removed by dialysis against deionized water at 25°C. As shown in Table 2, the particle size of the nanoparticles is about 90 nm, the particle size distribution is less than 0.2, the surface charge is about -20 mV, and the drug loading is about 70%. The particle size and morphology of the above PEG modified tetra-sulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles were determined by transmission electron microscopy, and the results are shown in Table 2 and Figure 1. Figure 6 The transmission electron microscopy image shows that the drug-loaded nanoparticles are uniform spherical.

[0094] Table 2 Particle size, particle size distribution, surface charge and drug loading of tetra-sulfide bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0095]

[0096] Example 7: Colloidal stability test of PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0097] (1) Storage stability of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0098] The PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) were subjected to colloidal stability test; the long-term storage stability of the prodrug self-assembled nanoparticles at 25°C was investigated with particle size change as an index. The results are shown in Table 6a. Figure 7 As shown in Table 6a, the prepared PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles had no obvious change in particle size and PdI after being placed at 25°C for 4 weeks, and had good long-term storage stability.

[0099] (2) Plasma stability of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0100] The PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) were subjected to plasma stability test; the PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles were diluted 20 times with rat plasma, incubated at 37°C for 60 h, and the particle size change thereof was determined by dynamic light scattering method at predetermined time points (0, 12, 24, 36, 48 and 60 h). The results are shown in Table 6b. Figure 7 As shown in Table 6b, the colloidal stability of γ-4S-2CTX nanoparticles, δ-4S-2CTX nanoparticles and γ-3S-2CTX nanoparticles was the best, and the particle size had no obvious change within 60 h. In contrast, the colloidal stability of γ-2S-2CTX nanoparticles was poor, and the nanoparticles had obvious change and uneven particle size distribution with the prolongation of incubation time.

[0101] Example 8: In vitro release test of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0102] (1) Release of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles in blank medium and under oxidation condition

[0103] The in vitro release of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in blank medium and oxidation condition was investigated with phosphate buffer solution (PBS) containing 30% ethanol at pH 7.4 as the release medium. 1 mL of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) (the cabazitaxel content was 200 μg / mL) was added to 30 mL of the release medium, and hydrogen peroxide (H2O2, 10 mM) was added to the release medium for the release under oxidation condition. At 37°C, samples were taken at the set time points, and the concentration of the released docetaxel was determined by high performance liquid chromatography to investigate the in vitro release of the nanoparticles in blank medium and oxidation condition. The results are shown in Table 1. Figure 8 As shown in Table 1a, the four-sulfur bond bridged cabazitaxel dimer self-assembled nanoparticles had good stability in the release medium, and only a small amount of the parent drug was released. Since the prodrugs in this example all had long aliphatic chains, the hydrophobicity of the prodrugs was increased, and, as shown in Table 1b, the hydrophobicity of the prodrugs was also enhanced with the increase in the number of sulfur atoms in the four-sulfur bond, so that the oxidation responsiveness of the four-sulfur bond bridged cabazitaxel dimer self-assembled nanoparticles was weak, and the most sensitive γ-3S-2CTX nanoparticles released only about 30% of CTX under 10 mM H2O2. Figure 8

[0104] (2) Release of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under reduction condition

[0105] The release of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles under reduction condition was investigated by replacing hydrogen peroxide (H2O2, 10 mM) with glutathione (GSH, 1 mM, 500 μM), cysteine (Cys, 1 mM, 500 μM) and ascorbic acid (AsA, 20 mM) by the preparation method of Example 8(1). The results are shown in Table 1c-g. Figure 8 As shown in Table 1c-g, the four-sulfur bond bridged dimer self-assembled nanoparticles had reduction-responsive release characteristics, and the reduction sensitivity order was γ-4S-2CTX nanoparticles > γ-3S-2CTX nanoparticles > δ-4S-2CTX nanoparticles > γ-2S-2CTX nanoparticles. Moreover, the four-sulfur bond bridged and the three-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles had super strong response to various reducing agents, and in the presence of Cys, the four-sulfur bond bridged and the three-sulfur bond bridged cabazitaxel dimer prodrug could release CTX faster and more.

[0106] Example 9: Pharmacokinetic study of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0107] ​SD rats weighing 200-250 g were randomly divided into groups and fasted for 12 h before administration, with free access to water. Cabazitaxel solution and the tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) were administered intravenously, respectively. The cabazitaxel dose was 4 mg / kg. Blood was collected from the orbital cavity at specified time points, and plasma was obtained. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0108] Experimental results are as follows Figure 9 As shown, cabazitaxel solution has a short half-life, and the cabazitaxel in it is rapidly cleared from the blood. In contrast, the circulation time of self-assembled nanoparticles of the tetrasulfide-bridged cabazitaxel dimer prodrug is significantly prolonged. Furthermore, different chemical linkages significantly affect the pharmacokinetic behavior of the self-assembled dimer prodrug nanoparticles. δ-4S-2CTX nanoparticles and γ-4S-2CTX nanoparticles, due to their strongest assembly stability and enhanced chemical stability in oxidized plasma environments, exhibit significantly prolonged circulation times in the blood, as shown in Table 3, with their total plasma concentration-time area under the curve (AUC) increasing to 460 times and 210 times that of the cabazitaxel solution, respectively. The total AUC of γ-3S-2CTX nanoparticles and γ-2S-2CTX nanoparticles is also significantly increased, being 171 times and 163 times that of the cabazitaxel solution, respectively.

[0109] Table 3. Pharmacokinetic parameters of tetrasulfide-bridged carbamate dimer prodrug self-assembled nanoparticles

[0110]

[0111] Example 10: Biodistribution of tetrasulfide-bridged carbamate dimer prodrug self-assembled nanoparticles

[0112] (1) Biodistribution of tetrasulfide-bridged carbamate dimer prodrug self-assembled nanoparticles

[0113] 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 500 mm, 3 At that time, the mice were injected via tail vein with a cabazitaxel solution and the PEG-modified tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6 (2), at a dose of 10 mg / kg. Mice were sacrificed at 1, 4, 8, and 24 h later, and the major organs (heart, liver, spleen, lung, kidney) and tumors were isolated. The drug concentration in plasma was determined by liquid chromatography-mass spectrometry.

[0114] (2) Biodistribution of DiR-loaded tetrasulfide-bridged carbamate dimer prodrug self-assembled nanoparticles

[0115] 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 500 mm,3 At the same time, cabazitaxel solution and free DiR and DiR-labeled prodrug self-assembled nanoparticles were injected into the tail vein of the nude mice, and the dosage of DiR was 1 mg / kg. After 4 or 24 hours, the nude mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney) and tumors were separated and analyzed by an in vivo imaging instrument.

[0116] The results are shown in Figure 10 As shown in

[0117] Example 11: Cellular uptake of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0118] The cellular uptake of small molecule prodrug self-assembled nanoparticles in mouse breast cancer (4T1) cells was determined by flow cytometry. 4T1 cells were seeded in a 12-well plate at a density of 5000 cells / mL and incubated in an incubator for 24 h to adhere. After the cells adhered, free coumarin-6 or coumarin-6-labeled tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles were added. The concentration of coumarin-6 was 200 ng / mL. After incubation at 37 °C for 0.5 h or 2 h, the cells were washed, collected and dispersed in PBS, and the cellular uptake of coumarin-6 was determined by flow cytometry.

[0119] The experimental results are shown in Figure 11 As shown in

[0120] Example 12: Cytotoxicity of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0121] (1) Cytotoxicity of tetrasulfide-bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0122] The toxicity of the four disulfide bond bridged dimer prodrug self-assembled nanoparticles on four kinds of tumor cells and one normal cell: 4T1 cells, human oral epidermoid carcinoma cells (KB, Hela contaminated cell line) cells, mouse pancreatic cancer (Panc02) cells, mouse prostate cancer (RM-1) cells and human normal liver (L02) cells was investigated by MTT method. First, the well-shaped cells were digested and diluted to 10000 cells / mL with culture solution. After blowing evenly, 200 μL of cell suspension was added to each well of the 96-well plate and incubated in the incubator for 24 h to adhere. After the cells adhered, the culture medium was replaced with fresh culture medium containing different concentrations of cabazitaxel solution or the four disulfide bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6 (2). In this experiment, the preparation and dilution of drug solution and nanoparticle preparation were carried out using the culture solution of the corresponding cells, and sterile filtration was carried out using a 0.22 μm filter membrane. Each well of the test solution was added with 200 μL, and three parallel holes were set for each concentration. The control group, i.e. without adding the test solution, was supplemented with 200 μL of culture solution alone, and was incubated in the incubator with the cells. 48 h after drug addition, the 96-well plate was taken out, the old culture medium was discarded, and 100 μL of fresh culture medium containing MTT was added to each well (MTT was prepared as a 5 mg / mL solution, and was diluted with culture medium before use), and was incubated in the incubator for 4 h. After discarding the culture medium, the 96-well plate was inverted on filter paper to completely absorb the residual liquid, and 200 μL of DMSO was added to each well on the shaker for 10 min to dissolve the blue-purple crystalline material. Set A1 hole (containing only 200 μL DMSO) as the zero hole. The absorbance value of each hole after zero setting was measured at 570 nm using an enzyme marker.

[0123] The results are shown in Table 2. Figure 12 As the prodrug needs to undergo an activation process to exert its effect in cells, the cytotoxicity of the four dimer prodrug nanoassemblies is weaker than that of the docetaxel solution. The cytotoxicity of the dimer prodrug nanoassemblies is closely related to their redox activation ability. The order of the anti-tumor activity of the four dimer prodrug nanoassemblies is: γ-4S-2CTX nanoparticles > γ-3S-2CTX nanoparticles > δ-4S-2CTX nanoparticles > γ-2S-2CTX nanoparticles. γ-4S-2CTX nanoparticles have super-sensitive reduction response ability and good cell uptake, and can effectively release CTX in tumor cells, so they show the strongest in vitro anti-tumor activity.

[0124] (2) Cytotoxicity of four disulfide bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with reducing agents

[0125] The toxicity of the four-sulfur bond bridged dimer prodrug self-assembled nanoparticles combined with reducing agents on RM-1 cells, 4T1 cells and L02 cells was investigated by MTT method. First, the cells with good morphology were digested and diluted to 10000 cells / mL with culture solution. After blowing evenly, 200 μL of cell suspension was added to each well of the 96-well plate and incubated in the incubator for 24 h to adhere. After the cells adhered, the culture medium was replaced with fresh culture medium containing different concentrations of cabazitaxel solution or the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2). In this experiment, the preparation and dilution of drug solution and nanoparticle preparation were carried out using the culture solution of the corresponding cells, and the sterile filtration was carried out using a 0.22 μm filter membrane. 100 μL of test solution was added to each well, and 3 parallel holes were set for each concentration. The control group, i.e. without adding the test solution, only supplemented with 200 μL of culture solution, was incubated in the incubator with the cells. 8 h after drug addition, 100 μL of Cys solution diluted to 1 mM and 2 mM with fresh culture medium was added to the 96-well plate of RM-1 cells; 100 μL of Cys and GSH solution diluted to 1 mM and 2 mM with fresh culture medium was added to the 96-well plate of 4T1 cells, as well as 1 mM, 2 mM and 40 mM of AsA solution; 100 μL of Cys and GSH solution diluted to 1 mM and 2 mM with fresh culture medium was added to the 96-well plate of L02 cells, as well as 40 mM of AsA solution. After 40 h of continuous culture, the 96-well plate was taken out, the old culture medium was discarded, and the 96-well plate was carefully rinsed with PBS for 2-3 times, then 100 μL of fresh culture medium containing MTT was added to each well (MTT was prepared as a 5 mg / mL solution, and was diluted with culture medium before use), and the plate was incubated in the incubator for 4 h. After discarding the culture medium, the 96-well plate was inverted on filter paper to completely absorb the residual liquid, and 200 μL of DMSO was added to each well to shake on the shaker for 10 min to dissolve the blue-purple crystalline material. Set A1 well (containing only 200 μL of DMSO) as the zero hole. The absorbance value of each well after zero setting was measured by enzyme marker at 570 nm.

[0126] The results are as follows Figure 12As shown, the combination of reducing agents significantly enhances the tumor cytotoxicity of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles, especially the cytotoxicity of the redox hypersensitive γ-4S-2CTX nanoparticles is improved; but the toxicity to normal cells only increases slightly, so the selective toxicity of the prodrug to tumor cells is further enhanced after combination. Among them, Cys can greatly improve the cytotoxicity of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles combined with the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles at a lower concentration due to its strongest reducing property; and AsA is reported to promote the production of H2O2 by tumor cells, induce oxidative stress, and induce apoptosis at high concentrations, and shows different degrees of improvement in cytotoxicity when combined with cabazitaxel solution and four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles, indicating that high concentrations of AsA have a potential mechanism of synergy with cabazitaxel chemotherapy.

[0127] Example 13: In vivo anti-tumor experiments of four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles

[0128] (1) Anti-tumor effect of four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in RM-1 model

[0129] Using RM-1 tumor-bearing C57BL / 6 mice as a model, the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) were administered via tail vein, and the cabazitaxel concentration was 2 mg / kg. When combined with reducing agents, 40 mg / kg of Cys solution was administered via tail vein 4 h after administration of cabazitaxel solution or prodrug self-assembled nanoparticles. Cabazitaxel solution group, Cys solution group and normal saline infusion group were set as control groups. The results are shown in Figure 13 As shown, γ-3S-2CTX nanoparticles and γ-4S-2CTX nanoparticles showed comparable tumor inhibition effect as cabazitaxel solution, and γ-4S-2CTX nanoparticles had obvious advantages in self-assembly ability, redox-sensitive release, blood circulation and cytotoxicity, and finally showed the strongest tumor inhibition activity. The anti-tumor effect of the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles after combination with Cys showed different degrees of enhancement. The anti-tumor effect of γ-4S-2CTX nanoparticles and γ-3S-2CTX nanoparticles was significantly enhanced after combination with Cys, and compared with cabazitaxel solution, the development of tumors was significantly delayed. Combination with Cys also greatly enhanced the anti-tumor effect of δ-4S-2CTX nanoparticles. In contrast, γ-2S-2CTX nanoparticles, which have relatively weak redox reactivity and poor self-assembly stability, do not show obvious enhancement in anti-tumor efficacy after combination with Cys. This phenomenon indicates that the Cys combination strategy is more suitable for four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles, which can achieve better results based on its better colloidal stability and redox sensitivity.

[0130] (2) Anti-tumor effect of four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles in 4T1 model

[0131] With 4T1 tumor-bearing BALB / c mice as a model, the four-sulfur bond bridged cabazitaxel dimer prodrug self-assembled nanoparticles prepared in Example 6(2) were administered by tail vein, with cabazitaxel concentration of 2 mg / kg and 4 mg / kg. When combined with reducing substances, 504 mg / kg of AsA solution was administered by tail vein 4 h after administration of cabazitaxel solution or prodrug self-assembled nanoparticles (cabazitaxel: 2 mg / kg), and cabazitaxel solution, and normal saline infusion groups were set as control groups. As shown in Figure 14 shown, γ-4S-2CTX nanoparticles still had good tumor inhibition effect on 4T1 tumor, and its therapeutic effect was equivalent to that of cabazitaxel solution at low-dose treatment (cabazitaxel: 2 mg / kg). At high-dose treatment (cabazitaxel: 4 mg / kg), γ-4S-2CTX nanoparticles significantly delayed tumor growth and controlled tumor volume at 400 mm 3 below, and did not show obvious toxicity. The mice in the cabazitaxel solution group had a sharp decrease in body weight after the first administration, indicating that cabazitaxel solution caused severe systemic toxicity. After combination with 504 mg / kg of AsA, γ-4S-2CTX nanoparticles (cabazitaxel: 4 mg / kg) showed the strongest anti-tumor effect. Due to the good colloidal stability and ultra-sensitive reduction response of γ-4S-2CTX nanoparticles, they have obvious advantages in pharmacokinetic behavior, tumor distribution, and cytotoxicity, which all ensure their good anti-tumor effect in vivo. At the same time, after combination with reducing AsA (504 mg / kg) which has certain tumor inhibition effect itself, the cell release of γ-4S-2CTX nanoparticles in tumor cells is further driven, and the anti-tumor effect is further enhanced.

[0132] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A tetrasulfide-bridged dimeric prodrug, characterized in that, The structural formula is as follows: or .

2. A method of preparing the tetrasulfide-bridged dimeric prodrug of claim 1, characterized in that, The method comprises the following steps: The binary acid containing a tetrasulfide bond is dissolved in a solvent and stirred uniformly; 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride are dissolved in a solvent and stirred uniformly, and then mixed with the binary acid solution containing a tetrasulfide bond; under N2 protection, the mixture is stirred at room temperature for 10-12 hours; 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride are added to the mixture, and the mixture is continuously stirred at room temperature for 12-24 hours under N2 protection; after separation and purification, the product is obtained; The binary acid containing a tetrasulfide bond is 4,4'-tetrasulfide dibutyric acid or 5,5'-tetrasulfide dipentanoic acid.

3. A tetrasulfide-bridged dimeric prodrug self-assembling nanoparticle, characterized in that, The PEG-modified small-molecule prodrug nanoparticle, the actively targeted small-molecule prodrug nanoparticle, the small-molecule prodrug nanoparticle loaded with a fluorescent substance or a hydrophobic drug; The PEG-modified small-molecule prodrug nanoparticle is a uniform nanoparticle spontaneously formed by dropping a mixed solution of the tetrasulfide bond-bridged dimer prodrug and a PEG modifier into water; the PEG modifier is TPGS, DSPE-PEG, PLGA-PEG or PE-PEG; the molecular weight of the PEG is 1000-5000; and the weight ratio of the tetrasulfide bond-bridged dimer prodrug to the PEG modifier is 90:10-70:

30. The active targeting small molecule prodrug nanoparticles are uniform nanoparticles spontaneously formed by adding a mixed solution of the tetrasulfide bridge dimer prodrug of claim 1 and a PEG modifier with a targeting head and a PEG modifier into water; the PEG modifier is TPGS, DSPE-PEG, PLGA-PEG or PE-PEG, the PEG modifier with a targeting head is DSPE-PEG-FA, DSPE-PEG-Biotin, cRGD-PEG-PLL, DSP-PEG 2k Pep-1 or DSPE-PEG-CREKA; the molecular weight of the PEG is 1000-5000; the weight ratio of the tetrasulfide bridge dimer prodrug to the PEG modifier is 90:10-70:30; The small-molecule prodrug nanoparticle loaded with a fluorescent substance or a hydrophobic drug is obtained by co-mixing the tetrasulfide bond-bridged dimer prodrug and the fluorescent substance or the hydrophobic drug and then self-assembling; the fluorescent substance is coumarin-6, rhodamine, DiR, DiI, Cy-5 or Cy-7; and the hydrophobic drug is camptothecin, hydroxycamptothecin or doxorubicin.

4. A pharmaceutical composition, characterized by, The tetrasulfide bond-bridged dimer prodrug comprises the tetrasulfide bond-bridged dimer prodrug and a pharmaceutically acceptable carrier or excipient.

5. Use of the tetrasulfide-bridged dimeric prodrug of claim 1 or the tetrasulfide-bridged dimeric prodrug self-assembled nanoparticle of claim 3 or the pharmaceutical composition of claim 4, characterized in that, The application in the preparation of a drug delivery system or the application in the preparation of an antitumor drug.

6. The tetrasulfide bond-bridged dimer prodrug of claim 1 or the tetrasulfide bond-bridged dimer prodrug self-assembled nanoparticle of claim 3 or the pharmaceutical composition of claim 4 in the preparation of an injection, oral administration or topical administration system.

7. Use of the tetrasulfide-bridged dimeric prodrug of claim 1 or the tetrasulfide-bridged dimeric prodrug self-assembled nanoparticle of claim 3 or the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating a tumor in combination with a reducing agent, characterized in that, The reducing agent is at least one of glutathione, cysteine, ascorbic acid, vitamin E, dithiothreitol, carotenoid or thioctic acid.

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