Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition and application thereof. The pharmaceutical composition comprises a reducing drug and reducing sensitive nanoparticles, the sensitive nanoparticles are reduced into cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, and the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles are reduced The cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles comprise the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles modified by PEG (Polyethylene Glycol) or modified by an active targeting group, or the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles entrapped with a hydrophobic fluorescent substance. According to the invention, the immune microenvironment is effectively reversed through the reducing drug, the permeation of the reduction-sensitive cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in tumors is enhanced, the active drug release of the reduction-sensitive nanoparticles is promoted, and the anti-tumor activity of the reduction-sensitive nanoparticles is comprehensively enhanced.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to a pharmaceutical composition and application thereof. Background Art

[0002] At present, the research focus of cancer treatment has shifted from tumor cells to tumor-specific microenvironment. Pathological abnormalities in the tumor microenvironment are the key to designing new nano-drug delivery systems. It is worth noting that the metabolic disorder in tumor cells turns to aerobic glycolysis to achieve rapid proliferation, resulting in a surge of ROS in mitochondria. In order to cope with the surge of ROS, tumor cells take up and synthesize a large amount of reducing substances, such as glutathione and cysteine, to maintain the survival of tumor cells. Therefore, compared with normal tissues and cells, the tumor microenvironment has redox characteristics.

[0003] Based on the abnormal redox properties in the tumor microenvironment, a large number of redox-sensitive nanodrug delivery systems have been designed and used for the intelligent delivery of therapeutic drugs. However, compared with normal cells, the redox properties of tumor cells are not obvious enough, resulting in the inability to fully release the therapeutic drugs in redox-sensitive nanodrugs, making it difficult for reduction-sensitive nanodrug delivery systems to achieve ideal anti-tumor efficacy. In addition, excessive oxidative stress in the tumor microenvironment can also limit the release of reduction-responsive drugs. This is because, on the one hand, high levels of ROS can also stimulate tumor cells to highly express transforming growth factor-β (TGF-β) and stimulate the activation of cancer-associated fibroblasts (CAFs). CAFs build a dense extracellular matrix (ECM) by secreting a large amount of collagen (α-SMA), which limits the penetration and uptake of nanodrugs; on the other hand, high levels of ROS can also promote regulatory T cells to release interfering cytokines, weakening CD8 + At the same time, ROS can also induce and recruit tumor-associated macrophages (TAMs), promote the recruitment and polarization of M2 macrophages, and thus lead to the formation of an immunosuppressive tumor microenvironment.

[0004] Therefore, regulating the redox state of tumor cells is of great significance in cancer treatment. It has been reported that reducing substances such as acetylcysteine, vitamin C, and reduced glutathione tend to accumulate in tumors, which is conducive to accelerating the activation of reduction-sensitive nanodrug delivery systems.

[0005] Although the above reducing agents have been used in the clinic to treat fibrotic diseases and combined with immunotherapy, there are currently no reports on the combined use of reduction-sensitive nanomedicines and reducing substances for anti-tumor treatment. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a pharmaceutical composition and its application, specifically relating to a reduction-sensitive cabazitaxel-Guerbet alcohol prodrug and the construction of self-assembled nanoparticles thereof, and the application of the composition in combination with a reducing drug in tumor treatment. By exploring the effects of different types of reducing drugs on the in vitro anti-tumor effect and drug release of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, the optimal drug combination is screened and obtained, thereby solving the problems of insufficient active drug release, poor permeability and immunosuppressive tumor microenvironment of reduction-sensitive nanomedicines in tumors.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] The invention discloses a pharmaceutical composition, comprising a reducing drug and reduction-sensitive nanoparticles; the reduction-sensitive nanoparticles are self-assembled nanoparticles of cabazitaxel-Guerbet alcohol prodrugs; the self-assembled nanoparticles of cabazitaxel-Guerbet alcohol prodrugs include PEG-modified or active targeting group-modified self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs, or self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs encapsulating hydrophobic fluorescent substances; the structural formula of the small molecule prodrug of cabazitaxel-Guerbet alcohol is:

[0009]

[0010] Optionally, the reducing drug includes one or more of vitamin C, reduced glutathione, acetylcysteine, and cysteine.

[0011] Optionally, the molar ratio of the reducing drug to the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is 1:(10-1000). Preferably, the molar ratio is 1:(100-500). More preferably, the molar ratio is 1:(200-500).

[0012] Optionally, the method for synthesizing the cabazitaxel-Guerbet alcohol small molecule prodrug comprises the following steps:

[0013] (1) dehydrating 2,2'-dithiodiacetic acid by acetic anhydride to obtain a dibasic acid anhydride;

[0014] (2) the Guerbet alcohol undergoes a ring-opening esterification reaction with the dibasic acid anhydride under the action of 4-dimethylaminopyridine to obtain an intermediate product, a Guerbet alcohol monoester;

[0015] (3) dissolving the intermediate product Guerbet alcohol ester in anhydrous dichloromethane, adding 1-hydroxybenzotriazole, carbodiimide and 4-dimethylaminopyridine dropwise thereto, activating in an ice bath, adding cabazitaxel to react, and purifying the reaction product by liquid phase separation to obtain the cabazitaxel-Guerbet alcohol small molecule prodrug;

[0016]

[0017] Optionally, in step (1), the dehydration reaction time is 0.5 h to 4 h; the dehydration reaction temperature is 15° C. to 37° C.; and the molar ratio of the 2,2'-dithiodiacetic acid to the acetic anhydride is 1:(1 to 50).

[0018] Optionally, in step (2), under nitrogen protection, the ring-opening esterification reaction time is 6h to 24h, the ring-opening esterification reaction temperature is 15°C to 37°C, and the intermediate product Guerbet alcohol monoester is obtained by separation through a chromatography column; the molar ratio of the 4-dimethylaminopyridine, the fatty alcohol and the dibasic acid anhydride is 1:(1-10):(1-10).

[0019] Optionally, in the step (3), the molar ratio of the intermediate product Guerbet alcohol monoester: the 1-hydroxybenzotriazole: the carbodiimide: the 4-dimethylaminopyridine: the cabazitaxel is 1:(1-10):(2-6):(0.2-5):(0.5-10); under nitrogen protection, the reaction temperature is 25°C to 37°C; and the reaction time is 24h to 48h.

[0020] Optionally, the method for preparing the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles comprises the following steps:

[0021] When the self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs are modified by PEG or active targeting groups, the preparation method comprises: mixing the cabazitaxel-Guerbet alcohol small molecule prodrugs with a PEG modifier or an active targeting modifier and then dripping the mixture into an aqueous solution to obtain the self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs modified by PEG or active targeting groups; wherein the PEG modifier comprises one of distearic acid phosphatidylethanolamine-methoxypolyethylene glycol, vitamin E polyethylene glycol succinate, distearic acid phosphatidylethanolamine-polyethylene glycol-folic acid and polylactic acid-co-glycolic acid-polyethylene glycol amphiphilic polymers or targeting groups; the active targeting modifier comprises one of antibodies, glycoproteins, lipoproteins, transferrin, polypeptides and folic acid;

[0022] When the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance, the preparation method comprises: mixing the hydrophobic fluorescent substance or drug, the PEG modifier and the cabazitaxel-Guerbet alcohol small molecule prodrug and then dropping the mixture into water to obtain the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles encapsulating the hydrophobic fluorescent substance; wherein the fluorescent substance comprises one of DiR, DiI, coumarin 6 and Cy5.5 fluorescent dyes.

[0023] Optionally, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles have a particle size of 50 nm to 200 nm, and a PDI of <0.2.

[0024] The present invention discloses an application of the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.

[0025] Optionally, the tumor includes one of breast cancer, lung cancer, prostate cancer, ovarian cancer, and melanoma.

[0026] Optionally, the application includes: first administering cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, and then administering a reducing drug after the nanoparticles reach maximum accumulation in the tumor; the time when the content of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in the tumor reaches a maximum is 0.5h to 12h; the time interval for administering the reducing drug is 0.5h to 8h; the administration amount of the reduction-sensitive cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is 1mg / kg to 10mg / kg; the administration amount of the reducing drug is 10mg / kg to 1000mg / kg.

[0027] Implementing the embodiments of the present invention will have the following beneficial effects:

[0028] The pharmaceutical composition disclosed in the present invention effectively reverses the immune microenvironment through reducing drugs, and while enhancing the penetration of reduction-sensitive cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in tumors, promotes the release of active drugs from reduction-sensitive nanoparticles, and comprehensively enhances the anti-tumor activity of reduction-sensitive nanoparticles. The specific advantages are:

[0029] (1) In the pharmaceutical composition provided by the present invention, the reducing drug can significantly improve the activation efficiency of reduction-sensitive nanoparticles in tumors, promote the release of active drugs, and increase the concentration of anti-tumor drugs in tumor cells.

[0030] (2) The pharmaceutical composition provided by the present invention, the reducing drug can effectively inhibit the activity of tumor-associated fibroblasts and reduce the content of matrix protein and collagen in the tumor, thereby enabling the reduction-sensitive nanoparticles to penetrate deep into the tumor.

[0031] (3) The pharmaceutical composition provided by the present invention, the reducing drug can also reverse the immune microenvironment, reduce the recruitment and differentiation of regulatory T cells and myeloid macrophages, promote T cell proliferation and killing effect, thereby enhancing the body's immune system's killing of tumor cells and providing conditions for combined use of immunotherapy.

[0032] (4) The pharmaceutical composition provided by the present invention is a reducing drug that is commonly used as an auxiliary drug in clinical chemotherapy and has good safety.

[0033] (5) The pharmaceutical composition provided by the present invention is administered with a reducing drug immediately after the reduction-sensitive cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles have reached maximum accumulation in the tumor. This administration method can ensure that the anti-tumor activity is significantly enhanced while avoiding the generation of additional toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the mass spectrum of the Cabazitaxel-Guerbet alcohol prodrug in Example 1 of the present invention.

[0035] Figure 2 is the cabazitaxel-Guerbet alcohol prodrug in Example 1 of the present invention 1 H-NMR spectrum.

[0036] Figure 3 This is a purity chart of the Cabazitaxel-Guerbet alcohol prodrug in Example 1 of the present invention.

[0037] Figure 4 This is a transmission electron microscopy image of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in Example 2 of the present invention.

[0038] Figure 5 : is the storage stability of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in Example 2 of the present invention; wherein A is a particle size change graph of the storage stability of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles at 37°C; B is a particle size change graph of the storage stability of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles at 4°C.

[0039] Figure 6 This is a cytotoxicity analysis of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution in Example 2 of the present invention combined with three reducing drugs; wherein A is the half inhibition concentration of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with reduced glutathione; B is the half inhibition concentration of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine; C is the half inhibition concentration of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with vitamin C.

[0040] Figure 7 This is a graph showing the in vitro release test of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with three reducing drugs in Example 2 of the present invention.

[0041] Figure 8 The tissue distribution of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution in Example 2 of the present invention; wherein A is the intratumoral accumulation of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution; and B is the cabazitaxel released from the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution in the tumor.

[0042] ns: P ≥ 0.05; *: P < 0.05; **: P < 0.01 (both two-sided t test)

[0043] Fig. 9 The tissue distribution of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution combined with acetylcysteine ​​in Example 2 of the present invention. A is the intratumoral accumulation diagram of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution combined with acetylcysteine; B is the cabazitaxel released from the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and the cabazitaxel solution combined with acetylcysteine ​​in the tumor.

[0044] ns: P ≥ 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001 (all two-sided t test)

[0045] Fig.10 The effect of continuous administration of acetylcysteine ​​on matrix protein and collagen. A is the effect of continuous administration of acetylcysteine ​​on matrix protein α-SMA; B is the effect of continuous administration of acetylcysteine ​​on collagen; C is the fluorescence quantitative column chart of the effect of continuous administration of acetylcysteine ​​on matrix protein α-SMA; D is the fluorescence quantitative column chart of the effect of continuous administration of acetylcysteine ​​on matrix protein collagen.

[0046] ns: P ≥ 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001 (all two-sided t test)

[0047] Fig.11 The effect of continuous administration of acetylcysteine ​​on the deep penetration of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles. A is the infrared imaging of DiR-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles; B is the fluorescence quantitative bar graph of DiR-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles; C is the fluorescence tissue section of DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles; D is the fluorescence quantitative bar graph of DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles. ns: P≥0.05; *: P<0.05; **: P<0.01; ***: P<0.001 (both two-sided t test)

[0048] Fig.12The present invention shows the anti-tumor effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the Balb / C mouse 4T1 breast cancer model according to Example 2 of the present invention; wherein A is the growth effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the Balb / C mouse 4T1 breast cancer model; B is the weight effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the Balb / C mouse 4T1 breast cancer model; C is the effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the tumor-bearing rate of the Balb / C mouse 4T1 breast cancer model.

[0049] ns: P ≥ 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001 (all two-sided t test)

[0050] Fig.13 is the effect of continuous administration of acetylcysteine ​​on the tumor immune microenvironment; A is the effect of continuous administration of acetylcysteine ​​on regulatory T cells; B is the effect of continuous administration of acetylcysteine ​​on CD4 + / CD8 + A is the effect of continuous administration of acetylcysteine ​​on T lymphocytes; C is the effect of continuous administration of acetylcysteine ​​on myeloid suppressor cells; D is the effect of continuous administration of acetylcysteine ​​on M1 / M2 macrophages.

[0051] Fig.14 The present invention shows the anti-tumor effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the C57 mouse B16F10 melanoma model; wherein A is the effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the growth of the C57 mouse B16F10 melanoma model; B is the effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the body weight of the C57 mouse B16F10 melanoma model; C is the effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​on the tumor-bearing rate of the C57 mouse B16F10 melanoma model.

[0052] ns: P ≥ 0.05; *: P < 0.05; **: P < 0.01; ***: P < 0.001 (all two-sided t test) DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0054] Example 1: Synthesis of Bataxel-Guerbet Alcohol Small Molecule Prodrug

[0055] Weigh 0.725g (4mmol) of 2,2'-dithiodiacetic acid and dissolve it in 15mL of acetic anhydride, and place it in a 50mL round-bottom flask. After it is completely dissolved, stir and react at 25℃ for 2h. After the reaction is completed, add a total of 3 times the volume of toluene in small amounts and multiple times, and remove toluene and acetic anhydride by vacuum distillation to obtain dithiodiacetic anhydride. Add an appropriate amount of anhydrous dichloromethane to dissolve the dithiodiacetic anhydride formed, and under stirring, slowly add 0.895g (3mmol) of Guerbet alcohol and 0.595g (2mmol) of 4-dimethylaminopyridine (DMAP) dissolved in anhydrous dichloromethane in sequence. React at room temperature for 12h, and monitor the reaction by thin layer chromatography. After the reaction, a crude product of Guerbet alcohol-dithiodiacetic acid monoester is obtained. The crude product is separated and purified by column chromatography using 3:1 cyclohexane-ethyl acetate (plus 0.1% glacial acetic acid) to obtain a pure product. 184.8 mg (0.4 mmol) of octyl lauryl alcohol-dithiodiacetic acid monoester was weighed and dissolved in dichloromethane, and 135 mg (0.7 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 84.8 mg (0.4 mmol) of 1-hydroxybenzotriazole, and 12.3 mg (0.10 mmol) of 4-dimethylaminopyridine dissolved in dichloromethane were added dropwise, and activated in an ice bath for 2 h. Then 292.8 mg (0.25 mmol) of cabazitaxel dissolved in dichloromethane was added, and the reaction was carried out at room temperature for 48 h. After the reaction, the target product was separated and purified by using a preparation liquid phase, and the mobile phase was acetonitrile to obtain a cabazitaxel-Guerbet alcohol small molecule prodrug.

[0056] Mass spectrometry was used to determine the structure of the prodrug in Example 1. The mass spectrum is shown in Figure 1 As shown, the mass spectrum results are MS (ESI) m / z [M+Na] + =1302.6428.

[0057] The structure of the prodrug in Example 1 was determined by H NMR spectroscopy. Figure 2 The results of H NMR spectrum analysis are as follows:

[0058] MS. 1 H-NMR (400MHz, DMSO-d6): δ7.97(d,J=7.2Hz,2H,Ar-H),7.85(d,

[0059] J=9.2Hz,1H,-NH-),7.74(t,J=7.3Hz,1H,Ar-H),7.66(t,J=7.5Hz,2H,Ar-H),7.42(t, J=7.6Hz,2H,Ar-H),7.37(d,J=7.4Hz,2H,Ar-H),7.17(t,J=7.1Hz,1H,Ar-H),5.81(t,J =8.8Hz,1H,40-H),5.06(t,J=8.5Hz,1H,-OH),4.94(d,J=10.0Hz,1H,10-H),4.68(s,1 H,19-H),4.48(s,1H,19-H),4.07-3.94(m,4H,COOCH2SSCH2COO),3.86(s,2H,GA20side chain OCH2),3.77-3.74(m,1H,11-H),3.73(s,2H,CH2COOCH2SS),3.58(d,J=7.0Hz,1H),3.32(s,3H,14-OCH3), ,3.27(s,3H,10-OCH3),2.24(s,3H,OOCCH3),1.79(s,3H,CH3),1.38(s,9H,C(CH3)3),1.31-1.17(m,32H,H atoms on the GA20 side chain),0.97(d,J=6.2Hz,6H,4-C(CH3)2),0.85(t,J=6.7Hz,6H,GA20 side chain CH3).

[0060] After high performance liquid chromatography analysis, the purity Figure 3 As shown, the purity of the cabazitaxel-Guerbet alcohol small molecule prodrug was 98.27%.

[0061] Example 2: Preparation of PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles

[0062] Accurately weigh DSPE-PEG 2k 1.5 mg of PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug 5 mg were dissolved and mixed with 1 mL of ethanol, and the ethanol solution was slowly added dropwise to 5 mL of deionized water under stirring, so that uniform PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles were spontaneously formed. The ethanol in the nanoparticles was removed by evaporation under reduced pressure.

[0063] The results are shown in Table 1. The particle size of the nanoparticles is about 100 nm, the particle size distribution is less than 0.2, the surface charge is about -20 mV, and the drug loading is about 50%. The particle size and morphology of the above-mentioned PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles were measured by transmission electron microscopy. The results are shown in Table 1. Figure 4 , Transmission electron microscopy images showed that the drug-loaded nanoparticles were uniform spherical.

[0064] Table 1 Particle size, particle size distribution, Zeta potential and drug loading of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles

[0065]

[0066] Example 3: Storage stability of PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles

[0067] The PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles prepared in Example 2 were subjected to a storage stability test; the long-term storage stability of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles at 37°C was tested using the particle size as an indicator. Figure 5 As shown, the prepared PEG-modified cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles had no significant changes in particle size and PDI after being placed at 4°C and 37°C for 3 weeks and 2 weeks, respectively, and had good storage stability.

[0068] Example 4: Cytotoxicity analysis of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and cabazitaxel solution combined with three reducing drugs.

[0069] The MTT method was used to investigate the toxicity of different reducing drugs combined with cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles on mouse breast cancer (4T1) cells. First, the cells with good morphology were digested, diluted to an appropriate concentration with fresh culture medium, and 2000 cells were added to each well of a 96-well plate after blowing evenly, and incubated in an incubator for 12 hours to make them adhere to the wall. After the cells adhered to the wall, the cabazitaxel solution and the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles prepared in Example 2 were added respectively. The cell culture medium was used for gradient dilution, 200 μL of the test solution was added to each well, and 3 parallel wells were used for each concentration. At 4 hours after the addition of the drug, acetylcysteine, vitamin C and reduced glutathione were gradiently diluted with cell culture medium, 50 μL of the test solution was added to each well, and 3 parallel wells were used for each concentration. After 48 hours, the 96-well plate was taken out, the old culture medium was poured out, and 135 μL of MTT solution diluted with fresh culture medium was added to each well (100 μL of fresh culture medium + 35 μL of 5 mg / mL MTT solution). After incubation in an incubator for 4 hours, the culture medium was discarded, and the 96-well plate was inverted on filter paper to fully absorb the residual liquid. After that, 200 μL of DMSO was added to each well and shaken on an oscillator for 10 minutes to dissolve the blue-purple crystals. The absorbance value was measured at 490 nm using an enzyme-labeled instrument.

[0070] The results are as follows Figure 6 As shown in Tables 2, 3, and 4, since the prodrug needs to undergo the process of smart drug release to play a role in the cell, the cytotoxicity of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles of Example 2 is weaker than that of the cabazitaxel solution. As shown in Tables 2-4, after the addition of the reducing drug, the drug release rate is accelerated, causing the cytotoxicity of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles to increase. Among them, as the concentration of the reducing agent increases, the cytotoxicity gradually increases. When the ratio of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles to the three reducing drugs is 1:200 and 1:500, the cytotoxicity of the prodrug self-assembled nanoparticles is the strongest. When the reducing agent ratio is increased to 1:1000, the cytotoxicity of the prodrug self-assembled nanoparticles is weakened instead. Therefore, 1:200 and 1:500 are the most suitable combination ratios of reducing drugs and cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, and 1:200 is used for subsequent experiments. At a ratio of 1:200, acetylcysteine ​​and reduced glutathione had comparable cytotoxic enhancement abilities on cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, while vitamin C had the weakest cytotoxic enhancement ability on cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles.

[0071] Table 2 Half inhibitory concentration (nmol / L) of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and cabazitaxel solution combined with reduced glutathione

[0072]

[0073] Table 3 Half inhibitory concentration (nmol / L) of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and cabazitaxel solution combined with acetylcysteine

[0074]

[0075] Table 4 Half inhibition concentration (nmol / L) of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and cabazitaxel solution combined with vitamin C

[0076]

[0077]

[0078] Example 5: In vitro release of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with three reducing drugs.

[0079] 0.250 μmol of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles prepared in Example 2 was added to 30 mL of PBS (pH 7.4) containing 30% ethanol (v / v). Blank medium, 1.666 mM reduced glutathione, 1.666 mM acetylcysteine ​​and 1.666 mM vitamin C were set respectively, and samples were taken at 1, 2h, 4h, 6h, 8h, 12h, and 24h, and the concentration of cabazitaxel was determined using a high performance liquid chromatograph.

[0080] The results are as follows Figure 7 As shown, in the blank medium, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles prepared in Example 2 only release a small amount of parent drug, indicating that the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles will not release a large amount of cabazitaxel in normal tissues or systemic circulation, thereby reducing systemic toxicity. And further investigated the reduction response release of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles under the stimulation of different reducing drugs (reduced glutathione, acetylcysteine ​​and vitamin C). Among them, the cabazitaxel release of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles under the stimulation of reduced glutathione and acetylcysteine ​​is significantly increased. And under the stimulation of vitamin C, cabazitaxel release is less, which is consistent with the cytotoxicity results, indicating that the combined use of reduced glutathione and acetylcysteine ​​can promote more cabazitaxel release. In addition, since reduced glutathione triggers drug release too fast, acetylcysteine ​​with a milder release rate is selected for subsequent research.

[0081] Example 6: Intratumoral accumulation of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles and cabazitaxel solution

[0082] Take 100 μL of mouse breast cancer cell suspension (4T1, 3x10 7cells / mL) were inoculated subcutaneously on the right back of female Balb / c mice to establish a mouse breast cancer ectopic tumor model. When the tumor on the back of the tumor-bearing mouse grew to 150-200mm 3 When the mice were treated with cabazitaxel solution (4 mg / kg) and C-GA prodrug self-assembled nanoparticles (4 mg / kg), the mice were randomly divided into 12 groups, each with 3 mice: cabazitaxel solution (4 mg / kg) and C-GA prodrug self-assembled nanoparticles (4 mg / kg). After drug administration, the tumor-bearing mice were killed at the specified time (0.5h, 1h, 2h, 4h, 8h, 12h), the tumor tissue was removed, the tumor tissue was homogenized and the sample was treated with acetonitrile precipitation method, and the prodrug and mother drug in the tumor tissue were determined by LC-MS to determine the maximum accumulation point of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in the tumor.

[0083] The results are as follows Figure 8 As shown, the experimental results show that in tumor tissue, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles have a significant tumor accumulation ability, which is significantly higher than the cabazitaxel solution, and 2 hours is the maximum accumulation time of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in tumor tissue. It is worth noting that although the tumor accumulation ability of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is significantly enhanced, the active drug cabazitaxel released is not as good as the cabazitaxel solution, which may limit the therapeutic effect of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles.

[0084] Example 7: Intratumoral accumulation of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles cabazitaxel solution combined with acetylcysteine

[0085] After the tumor-bearing mouse model was established according to Example 6, the tumor volume on the back of the tumor-bearing mouse grew to 300-400 mm 3 Around 20 days, the mice were randomly divided into 20 groups, 3 mice in each group: cabazitaxel solution (4 mg / kg), cabazitaxel solution (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg), cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg), cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg). After administration of cabazitaxel solution or cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, acetylcysteine ​​was injected 1.5 hours later, and the tumor-bearing mice were killed at the specified time (2h, 4h, 6h, 8h, 12h), the tumor tissue was removed, and the tumor tissue was homogenized and treated with acetonitrile precipitation method. The prodrug and mother drug in the tumor tissue were determined by LC-MS to determine whether the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​would promote the release of the active drug cabazitaxel.

[0086] The results are as follows Fig. 9As shown, the experimental results show that after the combination with acetylcysteine, the accumulation ability of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in the tumor has not decreased. In addition, after the combination with acetylcysteine, the activation of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles can be accelerated, and the release amount of active drug cabazitaxel at 6h, 8h, and 12h is significantly increased, even higher than the cabazitaxel released by the cabazitaxel solution, which is conducive to the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles to exert stronger anti-tumor activity.

[0087] Example 8: Effects of continuous administration of acetylcysteine ​​on matrix proteins and collagen.

[0088] After the tumor-bearing mouse model was established in Example 6, 390 mg / kg acetylcysteine ​​was administered to the tumor-bearing mice every 3 days for a total of 4 times. At the same time, glucose injection was injected at the same dose and number of days as a control. The tumor was then excised and subjected to Masson staining and α-SMA immunofluorescence staining to observe the improvement of the matrix protein and collagen of the tumor.

[0089] The results are as follows Fig.10 As shown, after continuous administration of acetylcysteine, the collagen and matrix proteins in the tumor tissue were significantly reduced compared with the glucose injection group, indicating that acetylcysteine ​​can significantly reduce the dense matrix in the tumor.

[0090] Example 9: Effect of continuous administration of acetylcysteine ​​on deep penetration of cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles.

[0091] After the tumor-bearing mouse model in Example 6 was established, 390 mg / kg acetylcysteine ​​was given to the tumor-bearing mice every 3 days, for a total of 4 times. At the same time, glucose injection was injected as a control according to the same dose and number of days. Afterwards, the tumor-bearing mice in the acetylcysteine ​​group were injected with DiR and DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg), and the tumor-bearing mice in the glucose group were injected with DiR and DiI, DiR and DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg). After 8 hours, the tumor was excised, and infrared in vivo imaging and fluorescent tissue sectioning were performed to observe the penetration of nanoparticles.

[0092] The results are as follows Fig.11As shown, the tumor accumulation was explored by injecting DiR and DiR-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles. After continuous administration of acetylcysteine, the tumor accumulation of DiR-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles was significantly enhanced. The tumor penetration was explored by injecting DiI and DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles. After continuous administration of acetylcysteine, the penetration ability of DiI-encapsulated cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles was significantly enhanced, and they could penetrate from the vicinity of blood vessels to the deep part of the tumor. Without the administration of acetylcysteine, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles could only accumulate near the blood vessels.

[0093] Example 9: In vivo anti-tumor experiment of acetylcysteine ​​combined with cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle therapy

[0094] After the tumor-bearing mouse model in Example 6 was established, the tumor volume grew to about 100-150 mm 3 ,The tumor-bearing mice were randomly divided into 6 groups, 5 mice in each group: glucose injection group, acetylcysteine ​​group (390 mg / kg), cabazitaxel solution group (4 mg / kg), cabazitaxel solution (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg) group, cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle group (4 mg / kg) and cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg) group. After the administration of cabazitaxel solution or cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, acetylcysteine ​​was injected 1.5 hours later, and the drug was given once every 3 days for a total of 4 times. The tumor volume (long diameter*short diameter*short diameter / 2) and body weight of tumor-bearing mice were measured and recorded every day, and the curves of tumor volume and body weight changes over time were drawn. After the experiment, the mice were killed, the tumor tissues were removed, and the tumor-bearing rate was calculated by weighing (tumor weight / mouse body weight*100%).

[0095] The results are as follows Fig.12As shown, although the cabazitaxel solution significantly inhibited the growth of tumors, since the cabazitaxel solution used a large amount of Tween 80 and ethanol as solubilizers, it was easy to cause severe allergic reactions, resulting in a rapid decrease in the weight of mice after administration, indicating that the cabazitaxel solution was highly toxic and had a narrow therapeutic window. The weight of mice in the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle group remained stable. However, it is worth noting that the tumors in the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle treatment group still grew rapidly, which was inferior to the cabazitaxel solution. After the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles were combined with acetylcysteine, their anti-tumor activity was significantly enhanced, and the tumor volume and tumor-bearing rate were significantly reduced, even better than the cabazitaxel solution. In addition, the weight of mice in the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​group did not decrease significantly, indicating that this drug combination has good safety.

[0096] Example 10: Effect of continuous administration of acetylcysteine ​​on the tumor immune microenvironment

[0097] Take 100 μL of cell suspension containing 3 million mouse melanoma cells (B16F10, 3x10 7 cells / mL) were inoculated subcutaneously on the right back of male C57 mice to establish a mouse melanoma ectopic tumor model. When the tumor volume grew to about 100-150mm 3 The tumor-bearing mice were given 390 mg / kg acetylcysteine ​​every 2 days for 4 times. At the same time, glucose injection was injected at the same dose and number of days as a control. After the end, the tumor was removed and the tumor-infiltrating CD4 + / CD8 + T lymphocytes, regulatory T cells (Treg), MDSC cells and M1 / M2 macrophages were measured.

[0098] The results are as follows Fig.13 As shown, after continuous administration of acetylcysteine, CD4 + / CD8 + The number of T lymphocytes increased from 0.494% / 1.697% to 2.473% / 4.837%, and the number of M1 pro-inflammatory macrophages increased from 5.529% to 17.576%, while the number of M2 suppressive macrophages (7.736%→0.583%), Treg cells (11.254%→4.208%), and MDSC cells (19.353%→8.376%) all decreased significantly, which is beneficial to reversing the immunosuppressive microenvironment in the tumor and providing a basis for combined immunotherapy to exert a stronger immune response.

[0099] Example 11: In vivo anti-tumor experiment of acetylcysteine ​​combined with Anti-PD1 and cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticle therapy

[0100] After the tumor-bearing mouse model in Example 4 was established, the tumor volume grew to about 100-150 mm 3 The tumor-bearing mice were randomly divided into 8 groups, with 5 mice in each group: glucose injection group, acetylcysteine ​​group (390 mg / kg), Anti-PD1 group (30 μg / mouse), Anti-PD1 (30 μg / mouse) combined with acetylcysteine ​​(390 mg / kg) group, cabazitaxel solution group (4 mg / kg), cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles group (4 mg / kg), cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg) group, and cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles (4 mg / kg) combined with acetylcysteine ​​(390 mg / kg) and Anti-PD1 (30 μg / mouse) group. After the administration of cabazitaxel solution or cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles, acetylcysteine ​​was injected 1.5 hours later, and Anti-PD1 was administered 2 hours later. The drug was administered once every 2 days for a total of 5 times. The tumor volume (long diameter * short diameter * short diameter / 2) and body weight of tumor-bearing mice were measured and recorded every day, and the curves of tumor volume and body weight over time were drawn. After the experiment, the mice were killed, the tumor tissue was peeled off, and the tumor-bearing rate was calculated by weighing (tumor weight / mouse weight*100%).

[0101] The results are as follows Fig.14 As shown in the figure, due to the immune-improving function of acetylcysteine, acetylcysteine ​​has certain antitumor activity compared with glucose injection. However, melanoma, as a rapidly proliferating malignant tumor, can achieve immune escape in many ways, such as upregulation of PD-L1. After combined with Anti-PD1, its antitumor activity is significantly enhanced. In addition, many traditional chemotherapy drugs are used in combination with Anti-PD1 in clinical practice to achieve the dual advantages of chemotherapy and immunotherapy. However, traditional chemotherapy causes severe systemic toxicity and weight loss, which makes it difficult for immunotherapy to work, so the therapeutic effect is minimal. After the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​not only showed a stronger antitumor effect than the cabazitaxel solution, but also did not cause weight loss in mice. At the same time, the combination of acetylcysteine ​​can reverse the immunosuppressive tumor microenvironment, which makes the combination of chemotherapy and immunotherapy possible. Therefore, without damaging the weight of mice, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles combined with acetylcysteine ​​and Anti-PD1 showed the strongest antitumor activity.

[0102] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A pharmaceutical composition, characterized in that Including reducing drugs and reducing-sensitive nanoparticles; The reduction-sensitive nanoparticles are self-assembled nanoparticles of cabazitaxel-Guerbet alcohol prodrug; The cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles include cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles modified with PEG or active targeting groups, or cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances; The Cabazitaxel-Guerbet alcohol small molecule prodrug structural formula is:

2. The pharmaceutical composition according to claim 1, characterized in that The reducing drug includes one or more of vitamin C, reduced glutathione, acetylcysteine ​​and cysteine.

3. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the reducing drug to the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is 1:(10-1000).

4. The pharmaceutical composition according to claim 1, characterized in that The synthesis method of the cabazitaxel-Guerbet alcohol small molecule prodrug comprises the following steps: (1) dehydrating 2,2'-dithiodiacetic acid by acetic anhydride to obtain a dibasic acid anhydride; (2) the Guerbet alcohol undergoes a ring-opening esterification reaction with the dibasic acid anhydride under the action of 4-dimethylaminopyridine to obtain an intermediate product, a Guerbet alcohol monoester; (3) dissolving the intermediate product Guerbet alcohol ester in anhydrous dichloromethane, adding 1-hydroxybenzotriazole, carbodiimide and 4-dimethylaminopyridine dropwise thereto, activating in an ice bath, adding cabazitaxel to react, and purifying the reaction product by liquid phase separation to obtain the cabazitaxel-Guerbet alcohol small molecule prodrug; 5. The pharmaceutical composition according to claim 4, characterized in that In step (1), the dehydration reaction time is 0.5h to 4h; the dehydration reaction temperature is 15°C to 37°C; the molar ratio of the 2,2'-dithiodiacetic acid to the acetic anhydride is 1:(1 to 50); In step (2), under nitrogen protection, the ring-opening esterification reaction is carried out for 6 hours to 24 hours, the temperature of the ring-opening esterification reaction is 15° C. to 37° C., and the intermediate product, Guerbet alcohol monoester, is obtained by separation through a chromatography column; the molar ratio of the 4-dimethylaminopyridine, the fatty alcohol and the dibasic acid anhydride is 1:(1-10):(1-10); In the step (3), the molar ratio of the intermediate product Guerbet alcohol monoester: the 1-hydroxybenzotriazole: the carbodiimide: the 4-dimethylaminopyridine: the cabazitaxel is 1:(1-10):(2-6):(0.2-5):(0.5-10); under nitrogen protection, the reaction temperature is 25°C to 37°C; and the reaction time is 24h to 48h.

6. The pharmaceutical composition according to claim 1, characterized in that The method for preparing the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles comprises the following steps: When the self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs are modified by PEG or active targeting groups, the preparation method comprises: mixing the cabazitaxel-Guerbet alcohol small molecule prodrugs with a PEG modifier or an active targeting modifier and then dripping the mixture into an aqueous solution to obtain the self-assembled nanoparticles of cabazitaxel-Guerbet alcohol small molecule prodrugs modified by PEG or active targeting groups; wherein the PEG modifier comprises one of distearic acid phosphatidylethanolamine-methoxypolyethylene glycol, vitamin E polyethylene glycol succinate, distearic acid phosphatidylethanolamine-polyethylene glycol-folic acid and polylactic acid-co-glycolic acid-polyethylene glycol amphiphilic polymers or targeting groups; the active targeting modifier comprises one of antibodies, glycoproteins, lipoproteins, transferrin, polypeptides and folic acid; When the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles encapsulating a hydrophobic fluorescent substance, the preparation method comprises: mixing the hydrophobic fluorescent substance or drug, the PEG modifier and the cabazitaxel-Guerbet alcohol small molecule prodrug and then dropping the mixture into water to obtain the cabazitaxel-Guerbet alcohol small molecule prodrug self-assembled nanoparticles encapsulating the hydrophobic fluorescent substance; wherein the fluorescent substance comprises one of DiR, DiI, coumarin 6 and Cy5.5 fluorescent dyes.

7. The pharmaceutical composition according to claim 1, characterized in that The particle size of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is 50nm-200nm, and the PDI is less than 0.

2.

8. Use of the pharmaceutical composition according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor includes one of breast cancer, lung cancer, prostate cancer, ovarian cancer, and melanoma.

10. The use according to claim 8, characterized in that: The applications include: First, the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles are administered, and after they reach maximum accumulation in the tumor, the reducing drug is administered; The time when the content of the cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles in the tumor reaches the maximum is 0.5h to 12h; The time interval for administering the reducing drug is 0.5h to 8h; The dosage of the reduction-sensitive cabazitaxel-Guerbet alcohol prodrug self-assembled nanoparticles is 1 mg / kg to 10 mg / kg; the dosage of the reducing drug is 10 mg / kg to 1000 mg / kg.

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