High-water-solubility paclitaxel compound chemically modified by diaconitic acid molecules as well as synthesis method and application of paclitaxel compound

Abstract

The invention discloses a high-water-solubility paclitaxel compound chemically modified by diaconitic acid molecules as well as a synthesis method and application of the paclitaxel compound chemically modified by the diaconitic acid molecules. The paclitaxel compound comprises paclitaxel and two aconitic acid molecules which are covalently bonded through ester bonds; the water-soluble paclitaxel compound is simple and convenient in preparation method and suitable for industrial production, paclitaxel drug molecules have amphiphilic properties, namely hydrophilicity and lipophilicity after being structurally modified by the method, the water solubility and the permeability are improved, the possibility of developing various oral and aerosol inhalation drug preparations is provided, and the compound is high in water solubility at normal temperature, low in cost and suitable for industrial production. According to the present invention, the prepared paclitaxel compound has characteristics of no foam generation during dissolution, more than 8 mg / ml, no need of any co-solvent, compound toxicity lower than the original drug paclitaxel, and ester bond accelerated hydrolysis fracture under the weak acid condition of the in vivo tumor tissue, such that the compound has advantages of certain tumor tissue targeting, good bioavailability and good anti-tumor effect.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis and medicine, and specifically relates to a highly water-soluble paclitaxel compound for treating malignant tumors, a preparation method thereof and the pharmaceutical use thereof as an anticancer drug. Background Art

[0002] Paclitaxel, also known as Taxol, has a unique mechanism for treating cancer and is now widely used as an anti-tumor drug for treating lung cancer, breast cancer, ovarian cancer and other cancers. However, the solubility of paclitaxel in water is very low, less than 0.03 mg / mL. In order to improve the water solubility of paclitaxel, the current clinical application of paclitaxel is its injection, which is a colorless viscous concentrated solution made of polyoxyethylene castor oil / anhydrous ethanol (ratio of 1:1) as a cosolvent. Although polyoxyethylene castor oil can increase the water solubility of paclitaxel, it can cause a variety of adverse reactions, such as allergic reactions, nephrotoxicity, neurotoxicity, cardiovascular toxicity, etc. In order to avoid severe allergic reactions, in clinical practice, corticosteroids, diphenhydramine and other drugs are often injected before paclitaxel injection, but some patients still have allergic reactions after paclitaxel injection. Abraxane, a solvent-free paclitaxel albumin nanoparticle suspension for injection, was approved by the FDA for marketing in the United States in 2005. Albumin-bound paclitaxel is a nanoparticle made of paclitaxel and human albumin by high-pressure vibration technology. Each bottle contains 100 mg of paclitaxel and about 900 mg of human albumin. Paclitaxel is the active ingredient of the drug, and human albumin is used as an excipient to disperse and stabilize the particles and carry the main drug. Paclitaxel albumin nanoparticles reduce toxic side effects and eliminate the toxic side effects caused by polyoxyethylene castor oil. There is no need to give patients anti-allergic drugs for pretreatment before infusion, and no special infusion equipment is required, which greatly shortens the infusion time. However, during the preparation process of albumin-bound paclitaxel, paclitaxel and human albumin are made into nanoparticles by high-pressure vibration technology. This process is only physical mixing, not covalently connecting paclitaxel to albumin, and there is a small amount of drug leakage. Before medication, the particles of albumin-bound paclitaxel are dissolved into a soluble solution because the albumin in the system is prone to foaming. Some patients have allergic reactions to albumin during the use of this drug. In addition, cancer patients with hypertension, heart disease, hypervolemic heart failure, severe anemia, etc. will have adverse reactions to the solubilizing agent albumin in this drug.

[0003] The existing methods for solving the water solubility of paclitaxel are divided into dosage form changes and chemical structure modifications. In recent years, there are mainly liposomes, water-soluble high molecular polymer carriers and albumin carriers. For example, Chinese patent CN104758250A discloses a paclitaxel liposome and its preparation method and use. The paclitaxel liposome prepared by this invention patent has a small particle size and a narrow distribution, which can significantly improve the penetration rate of liposome drugs, allowing the drug to penetrate into the lesion site more quickly for administration, thereby enhancing the efficacy. However, the liposome drug delivery system in this scheme has problems such as low encapsulation rate, drug leakage and stability. The scheme of water-soluble high molecular polymer carriers, such as Chinese patent CN106554329B, discloses a water-soluble paclitaxel anticancer drug compound and its preparation method and use. This invention patent technology covalently combines the anticancer drug active part paclitaxel with polyethylene glycol monoalkyl ether through a connecting group (carbonyl methoxyacetyl) to form the water-soluble paclitaxel anticancer drug compound. Chinese patent CN104231047B water-soluble targeted activated paclitaxel derivatives and their preparation and use use water-soluble polyamino acids as carriers to improve the water solubility of paclitaxel original drugs. However, these patents have complex structural modification methods, uneven molecular weight of water-soluble polymer carriers, and high requirements for separation and purification methods. In particular, patent CN104231047B uses a single cis-aconitic anhydride (CA) to modify various original drugs, through the ring-opening reaction between the original drug and CA, using triethylamine as a catalyst for synthesis, and further connected with N, N'-di-dodecyl-L-glutamic acid diamide, which improves the hydrophobicity of the drug. Chinese patent CN101658516B discloses a pharmaceutical composition of a paclitaxel drug and a pharmaceutically acceptable biological carrier and a preparation method thereof. The pharmaceutical composition of the paclitaxel drug and the carrier protein is a nanoparticle suspension prepared with a carrier protein, an organic phase, a stabilizer, a freeze-drying protective agent, and a paclitaxel drug. However, some patients are allergic to albumin, and albumin preparations do not have the diffusivity and penetration of small molecule drugs to tumor tissues, which limits the clinical application of the pharmaceutical composition prepared by this method. Chinese patent CN111281979A discloses a cationic polyphosphazene compound, a polyphosphazene-drug coupling compound and a preparation method thereof, which is obtained by chemically connecting a hydrophobic drug to a compound, such as connecting a spacer group to a cis-aconitic anhydride connecting group on the drug molecule docetaxel, but this method has problems such as uneven molecular weight, poor drug loading repeatability, and difficulty in completely determining the coupled molecular structure. Therefore, providing a paclitaxel small molecule compound has become a technical problem that needs to be solved in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a small molecule drug of a highly water-soluble bisaconitic acid-modified paclitaxel compound, a synthesis method and pharmaceutical use, and to solve the problems of stability, drug leakage and difficulty in purification in the existing liposome drug system.

[0005] Specifically, this application provides the following solutions:

[0006] First, the present application provides a highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound, the structure of which is shown in the following formula (I):

[0007]

[0008] Secondly, the present application provides a method for synthesizing the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I), the specific steps of which are as follows:

[0009] 1) mixing aconitic anhydride and paclitaxel in an anhydrous solvent, and subjecting the mixture to an esterification reaction at 90° C. for 8 hours to obtain a reaction product;

[0010] The molar mass ratio of the above-mentioned paclitaxel to aconitic anhydride is 1:5;

[0011] The above-mentioned anhydrous solvent is preferably pyridine;

[0012] 2) The reaction product obtained in step 1) was evaporated at 60° C. to remove as much anhydrous solvent as possible, and a dark brown viscous product was obtained, which was the evaporation product; after adding laboratory pure water and stirring (the viscous product was partially dissolved and partially precipitated), the mixture was centrifuged at 5000 rpm for 5 min, and the supernatant was collected;

[0013] The volume ratio of the viscous product to the added water is preferably 1:10;

[0014] In a specific implementation, the precipitate obtained by centrifugation can also be added with water solvent again, centrifuged and the supernatant is obtained;

[0015] 3) placing the supernatant obtained in step 2) into a 300 molecular weight dialysis bag and dialyzing with deionized water for 24 hours to remove impurities and obtain a dialyzed product; then freeze-drying at -20°C to obtain a powdery product;

[0016] 4) Recrystallization: The powdery product obtained in step 3) is dissolved in 60°C water (experimental grade), allowed to stand at 4°C for 2 hours, filtered, and the solid is dried to obtain the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound.

[0017] Third, the present application provides the use of the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I) in the preparation of anticancer drugs. The anticancer drug is a drug for preventing or treating solid tumor cancer (such as breast cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, colon cancer, rectal cancer, bladder cancer), sarcoma, glioma, and blood system cancer (such as leukemia, lymphoma, myeloma). The dosage form of the above anticancer drug includes oral, aerosol inhalation drug and other preparations.

[0018] Fourthly, the present application provides a pharmaceutical composition, which is obtained by dissolving the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I) in a pharmaceutically acceptable carrier or excipient, and the dosage form can be tablets (oral), aqueous solution, or spray; the carrier is preferably a liquid carrier, such as water, PBS buffer, physiological saline or glucose solution, etc., to prepare injections and various oral and aerosol inhalation pharmaceutical preparations.

[0019] The highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound (I) prepared in the present application contains a lipophilic anticancer drug active part and a hydrophilic part (carboxyl and hydroxyl), wherein the lipophilic anticancer drug active part is a drug compound molecule paclitaxel with anticancer activity, and the hydrophilic part is a compound formed by covalently bonding two molecules of water-soluble aconitic acid and an anticancer drug compound paclitaxel through an ester bond. After the paclitaxel drug molecule is structurally modified by this method, it has amphiphilic properties, i.e., hydrophilicity and lipophilicity, and its water solubility and permeability are improved, providing the possibility that the compound can be used to develop oral and atomized inhalation drug preparations.

[0020] Furthermore, the water-soluble modification of paclitaxel in the present application utilizes water-soluble groups including various organic acids and water-soluble amino acids. Such carriers can be further made into salts such as carboxylates, sulfonates, phosphates or ammonium salts after esterification with the hydroxyl groups of the original drug, which greatly improves the water solubility. Since paclitaxel has a large molecular weight and is mainly composed of hydrophobic groups in the molecule, it is generally difficult to significantly improve its solubility by inserting one or two hydrophilic groups such as amino or carboxyl groups. The present application selects aconitic anhydride, and after esterification of the 2- and '7-hydroxyl groups of paclitaxel, four water-soluble carboxyl groups are added to the paclitaxel molecule, and the four carboxyl groups can be made into sodium salts, further improving the water solubility. At the same time, in the esterification method of taxane compounds, the esterification reaction conditions are such as the acyl chloride method, and the conventional method is to directly carry out the esterification of acid and alcohol in the presence of a carbodiimide dehydrating agent such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), etc. and a catalyst 4-dimethylaminopyridine (DMAP). However, these methods will bring problems of subsequent product separation and purification. Therefore, the present application uses aconitic anhydride to react with paclitaxel in pyridine at 90-95°C to esterify the two hydroxyl groups of paclitaxel. The reaction is simple and the product purification process is simple.

[0021] The method for preparing the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound provided by the present invention is simple, rapid, and has a high yield, and is suitable for large-scale industrial production. In the body, cis-aconitic acid is an intermediate product of citric acid isomerized to generate isocitric acid under the action of aconitase (located in the mitochondria), and can be degraded and absorbed in the body. Compared with the prior art (such as Chinese patent CN101658516B), the bisaconitic acid molecule modified paclitaxel compound prepared by the present method is a single small molecule compound, does not contain carrier protein, organic phase, stabilizer, freeze-drying protective agent, and the indicators such as drug purity and quality are controllable, the physical and chemical properties are more stable, and the preparation process omits the high-pressure homogenization technology of the above-mentioned patent, thereby being simpler and more feasible, and more suitable for mass preparation and industrial production. The water solubility at room temperature reaches more than 8 mg / ml (saturated dissolution), the effective drug concentration is much higher than the percentage concentration of 0.075-1.0% in patent CN101658516B, and the compound can be dissolved without any cosolvent. When the compound is dissolved in an aqueous solution, it is a clear aqueous solution. Since tumor tissue cells produce a large amount of lactic acid through anaerobic metabolic glycolysis, the pH value of the tumor tissue microenvironment is 5.7-7.2, which is significantly lower than the pH value of 7.4 of normal tissue (Webb BA, Chimenti M, Jacobson MP, et al. Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer, 2011, 11: 671-677.). The bisaconitic acid molecule modified paclitaxel compound prepared by the method accelerates the hydrolysis and cleavage of the ester bond under the weakly acidic conditions of tumor tissue in vivo, releases the original drug paclitaxel molecules and enriches in the tumor tissue, so the compound has certain tumor tissue targeting, good bioavailability and anti-tumor effect.

[0022] Compared with the above-mentioned prior art, the method for preparing the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound obtained by the present invention has the advantages of being simple, rapid and having a high yield; after the structure of paclitaxel is modified by this method, the chemical properties are stable and the water solubility is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 H NMR spectrum of water-soluble paclitaxel anticancer drug compound.

[0024] Figure 2 Photograph of an 8 mg / mL solution of the water-soluble paclitaxel anticancer drug compound.

[0025] Figure 3 HPLC analysis results of purified water-soluble paclitaxel anticancer drug compound.

[0026] Figure 4 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human breast cancer MCF7 cells.

[0027] Figure 5 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human non-small cell lung cancer A549 cells.

[0028] Figure 6 Experimental diagram of water-soluble paclitaxel anticancer drug compound on human umbilical vein endothelial HUVEC cells.

[0029] Figure 7 Experimental diagram of water-soluble paclitaxel anticancer drug compound on osteogenic MC3T3 cells.

[0030] Figure 8 Photo of tumor growth of human breast cancer cell MCF-7 xenografts in nude mice without drug treatment.

[0031] Fig. 9 Photo of the inhibitory effect of the common paclitaxel anticancer drug compound on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice.

[0032] Fig.10 Photo showing the inhibitory effect of water-soluble paclitaxel anticancer drug compound on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice.

[0033] Fig.11 Photo of the inhibitory effect of water-soluble paclitaxel anticancer drug compound on breast cancer cell 4T1 xenograft tumor growth in mice.

[0034] Fig.12 The inhibitory effect of water-soluble paclitaxel anticancer drug compound on breast cancer cell 4T1 and MCF-7 xenograft tumor growth in mice. Tumor volume versus time. DETAILED DESCRIPTION

[0035] In order to more completely introduce the characteristics of the present invention, the present invention is further introduced below in conjunction with specific implementation cases. For the water-soluble paclitaxel anticancer drug compound within the scope of the present invention, the same or similar method can be used to synthesize, and it has been verified that the same or similar results are obtained. The described embodiments are helpful for the understanding and implementation of the present invention and do not constitute a limitation of the present invention.

[0036] Example 1 Preparation of highly water-soluble bisaconitic acid-modified paclitaxel compounds

[0037] 1. Take 0.2135g of paclitaxel (purchased from Dalian Meilun Company, the same below) and 0.1951g of cis-aconitic anhydride (purchased from Aladdin Reagent Company, the same below), put them into a 60°C oven and dry them for half an hour for later use. In this embodiment, the molar mass ratio of paclitaxel to aconitic anhydride is 1:5.

[0038] 2. Take 1g of anhydrous calcium chloride and put it in a 90℃ oven to dry for half an hour (try to ensure it is dry as much as possible) and set aside.

[0039] 3. Take 5 ml of pyridine and place the anhydrous calcium chloride in step 2 in pyridine (C5H5N) for water absorption.

[0040] 4. Centrifuge the pyridine treated with water in step 3 at 5000 rpm for 5 minutes, take the supernatant and put it into a penicillin bottle, add a dry magnet and set aside.

[0041] 5. Dissolve the paclitaxel and aconitic anhydride dried in step 1 in the pyridine in step 4 and wait for reaction.

[0042] 6. Place the pyridine to be reacted and the raw materials in step 5 in a 90°C magnetic stirring heating mantle for reaction for 8 hours.

[0043] 7. The product after the reaction was treated with a rotary evaporator at 60°C for 1 hour to remove as much pyridine as possible to obtain a dark brown viscous product. 30 ml of laboratory pure water (purchased from Millipore Corporation) was added and stirred to dissolve to obtain a partially dissolved and partially precipitated product.

[0044] 8. Centrifuge the partially dissolved and partially precipitated product in step 7 at 5000 rpm for 5 min, filter, and take the supernatant for later use; the separated precipitate can be recovered and reused; put the supernatant into a 300 molecular weight dialysis bag (300 molecular weight grade of Spectrum, USA) and dialyze with deionized water.

[0045] 9. In step 8, the deionized water used for dialysis should be changed every two hours for 24 hours.

[0046] 10. The dialyzed product in step 9 was freeze-dried at -20°C for 24 hours to obtain a light yellow powder (i.e., a water-soluble paclitaxel compound) having the structural formula shown in the above formula (I). The yield was calculated to be 60% and the purity was 99%.

[0047] 11. Recrystallization: gradually add a small amount of experimental pure water (60° C.) to the light yellow powder obtained in step 10. After the powder is completely dissolved, cool it naturally to room temperature, put it in a refrigerator at 4° C. for 2 hours, filter it, and dry the solid (freeze-dry it at -20° C. for 24 hours) to obtain a purified sample, which is a highly water-soluble diaconitic acid molecule chemically modified paclitaxel compound after purification (purified water-soluble paclitaxel anticancer drug compound).

[0048] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the yellow powder in step 10, where the y-axis represents intensity.

[0049] Figure 2The light yellow powder obtained in step 10 above is dissolved in water to obtain a light yellow clear solution. The powder can be quickly dissolved in water without foam. The concentration of the obtained solution is 8 mg / ml, indicating that the compound has good water solubility.

[0050] Figure 3 This is the HPLC analysis chart of the purified water-soluble paclitaxel anticancer drug compound obtained in step 11 above. The sharp peaks in the chart prove that the synthesized compound has a single structure and high purity.

[0051] Example 2 Preparation of water-soluble paclitaxel anticancer drug compound

[0052] This embodiment includes an injection made of water, saline (0.9% sodium chloride) or glucose (5%) injection, and the preparation formula contains the water-soluble paclitaxel anticancer drug compound of the present invention, and the content of each component in the formula is calculated by weight percentage.

[0053] 1) Aqueous injection of water-soluble paclitaxel anticancer drug compound

[0054] 100 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (step 11) was added to a 20 mL volumetric flask, and the volume was adjusted to 20 mL with deionized water. The prepared injection solution was filtered through a 0.2 μm filter (Mabbio Biomembrane Technology (Nantong) Co., Ltd., model 0.2 μm, the same below), and then filled into a sterile glass bottle.

[0055] 2) 1.0% water-soluble paclitaxel anticancer drug compound in normal saline (0.9% sodium chloride) injection

[0056] 50 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (step 11) was added to a 10 mL volumetric flask, and the volume was made up to 100 mL with physiological saline. After shaking, the prepared injection solution was filtered through a 0.2 micron filter and then filled into a sterile glass bottle.

[0057] 3) 5% glucose injection of water-soluble paclitaxel anticancer drug compound

[0058] 50 mg of the purified water-soluble paclitaxel anticancer drug compound prepared in Example 1 (step 11) was placed in a 10 mL volumetric flask, and the volume was made up to 10 mL with 5% glucose injection solution. After shaking, the prepared injection solution was filtered through a 0.2 micron filter and then filled into a sterile glass bottle.

[0059] Example 3. Cytological test of the inhibitory effect of water-soluble paclitaxel on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice

[0060] Take tumor cells in logarithmic growth phase (human non-small cell lung cancer A549 cells, human breast cancer MCF-7 cells, human umbilical cord endothelial HUVEC normal cells and MC3T3 osteoblasts purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, all commercial cells), wait for the cells to cover the bottom of the culture bottle, digest the cells with 0.25% trypsin, and use culture medium to blow the cells into a single cell suspension. Count the cells using a cell counting plate, and the number of cells in each well is about 10 5 / mL was inoculated in a 96-well plate, and 200 μL of cell culture fluid (Gibco cell culture medium, the main components are amino acids, glucose, inorganic salts, vitamins and trace elements, etc.) was added to each well. After the 96-well plate was cultured in a cell culture incubator for 24 hours, an inverted microscope was used for observation. When the cells were evenly spread on the bottom of the plate, different concentrations of purified water-soluble paclitaxel (20nM, 100nM, 200nM, 500nM, 1μM, 5μM, 10μM, 15μM, 20μM) prepared in Example 1 (step 11) were prepared with PBS solution. It was added to each well in order from high to low. In order to increase the accuracy of the experiment, 4 wells were set for each concentration of the drug. The original drug paclitaxel (1 microliter of dimethyl sulfoxide DMSO solubilized) in the positive control group was also added at concentrations of (20nM, 100nM, 200nM, 500nM, 1μM, 5μM, 10μM, 15μM, 20μM). The blank control group was not added with drugs but only with an equal amount of cell culture fluid. After 24 hours, 100μL DMEM (RPMI) (containing 10% CCK-8) was added to each well, and the cells were placed in an incubator and continued to be cultured for 4 hours. The wavelength was selected as 492nm as the detection wavelength, and 630nm as the reference wavelength. The absorbance (OD) value of each well was measured using an enzyme marker (Thermo Fisher Multiskan FC). The data was recorded and repeated three times.

[0061] Calculation of tumor cell growth inhibition rate (IR) value:

[0062] IR (%) = (1 - average OD value of the experimental group / average OD value of the normal control group) * 100%, with drug concentration as the horizontal axis and cell inhibition rate as the vertical axis.

[0063] EXCEL data analysis software was used to analyze the data, and T-test and variance analysis were performed between groups. P < 0.05 indicated statistical significance.

[0064] Results and Discussion:

[0065] The inhibitory effect of the drug on tumor cells was evaluated using human non-small cell lung cancer A549 cells and human breast cancer MCF-7 cells.

[0066] like Figure 4As shown, the water-soluble paclitaxel drug has a good inhibitory effect on human non-small cell lung cancer A549 cells, but the inhibitory effect on A549 tumor cells is slightly lower than that of the positive control drug paclitaxel.

[0067] like Figure 5 As shown, at each concentration, water-soluble paclitaxel has a higher inhibition rate. The positive control drug paclitaxel (in the concentration range of 20nM to 20μM) has a stronger inhibitory effect on human breast cancer MCF-7 cells than water-soluble paclitaxel.

[0068] The inhibitory effect of the drug on normal tissue cells was evaluated using human umbilical cord endothelial HUVEC normal cells and MC3T3 osteoblasts.

[0069] like Figure 6 As shown, the positive control drug paclitaxel (in the concentration range of 20 nM to 20 μM) has a stronger inhibitory effect on normal human umbilical cord endothelial HUVEC cells than water-soluble paclitaxel. The drug toxicity of water-soluble paclitaxel is weaker than that of the original drug paclitaxel.

[0070] like Figure 7 As shown, at a concentration of 5 μM, the inhibitory effect of water-soluble paclitaxel on MC3T3 osteoblasts is basically equivalent to that of the positive control drug paclitaxel. However, at other drug concentrations, the positive control drug paclitaxel (within the concentration range of 20 nM to 15 μM) is more toxic to osteoblasts than water-soluble paclitaxel.

[0071] In summary, compared with the original drug paclitaxel, the anti-tumor efficacy of water-soluble paclitaxel is slightly reduced, but it still has a good anti-tumor effect, but the toxicity of the drug to normal tissue cells is reduced.

[0072] Example 4 Pharmacodynamics test of the inhibitory effect of water-soluble paclitaxel on the growth of human breast cancer cell MCF-7 xenograft tumors in nude mice

[0073] 1) Preparation of drug solution

[0074] The purified water-soluble paclitaxel prepared in Example 1 (step 11) was added to 0.9% sodium chloride solution to prepare a liquid with a concentration of 5 mg / ml, and the administration volume was 0.2 ml / 20 g, that is, the dosage was 10 mg / kg. It was then diluted with 0.9% sodium chloride solution to other required doses. The control drug paclitaxel injection (30 mg*5 ml / box, Haikou Pharmaceutical Factory) was fully diluted with physiological saline to 1 mg / ml, and the administration volume was 0.2 ml / 20 g, that is, the dosage was 10 mg / kg.

[0075] 2) Source, species, strain and grouping of experimental animals

[0076] BALB / c-null nude mice (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), weight: 18-24g, gender: female. Experimental animal groups: 6 blank controls, 6 paclitaxel controls, 6 water-soluble paclitaxel treatment groups

[0077] 3) Implanted tumor and experimental methods

[0078] The human breast cancer cell line MCF-7 was inoculated subcutaneously in the axilla of nude mice, with a cell inoculation volume of 2*10 6 Human breast cancer cell line MCF-7 in logarithmic growth phase was prepared into 1×10 8 / ml cell suspension, 0.1ml was inoculated into the right axilla of nude mice. The diameter of the transplanted tumor of nude mice was measured with a vernier caliper. When the tumor grew to 100-300mm 3 The animals were then randomly divided into groups. The anti-tumor effect of the test substance was dynamically observed by measuring the tumor diameter. The tumor diameter was measured once every 3 days. The administration volume was 0.5 ml / 20 g. After 28 days, the mice were killed and the tumor was surgically removed and weighed.

[0079] 4) Results and Discussion

[0080] Test results such as Figure 8 , Fig. 9 and Fig.10 As shown, the drug was injected into the tail vein at a dose of 10 mg / kg, once every 2 days, for a total of 14 times. The tumor inhibition rate of the experimental group on the human breast cancer cell MCF-7 nude mouse transplanted tumor reached 81.2%±0.8%. The original drug of paclitaxel was injected into the tail vein at a dose of 10 mg / kg, once every 2 days, for a total of 14 times, and the tumor inhibition rate of the human breast cancer cell MCF-7 nude mouse transplanted tumor reached 72.6%±0.8%. The test results show that the inhibition rate of the water-soluble paclitaxel drug 10 mg / kg on the tumor is 8.6% higher than that of the original drug paclitaxel 10 mg / kg in the positive control group, but the effect on the weight of the experimental mice is less, indicating that the water-soluble paclitaxel is more effective than paclitaxel and has lower toxicity.

[0081] Example 5 Pharmacodynamics and histological analysis of the inhibitory effect of water-soluble paclitaxel on the growth of breast cancer cell 4T1 xenograft tumors in mice

[0082] 1) Preparation of drug solution

[0083] The purified water-soluble paclitaxel prepared in Example 1 (step 11) was added to 0.9% sodium chloride solution to prepare a liquid with a concentration of 5 mg / ml, and the administration volume was 0.2 ml / 20 g, that is, the dosage was 10 mg / kg. It was then diluted with 0.9% sodium chloride solution to other required doses. The control drug paclitaxel injection (30 mg*5 ml / box, Haikou Pharmaceutical Factory) was fully diluted with physiological saline to 1 mg / ml, and the administration volume was 0.2 ml / 20 g, that is, the dosage was 10 mg / kg.

[0084] 2) Source, species, strain and grouping of experimental animals

[0085] BALB / c mice (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), weight: 18-24g, sex: female. Experimental animal groups: 6 blank controls, 6 paclitaxel controls, 6 water-soluble paclitaxel treatment groups 3) Tumor implantation and experimental methods

[0086] Human breast cancer cell line 4T1 was inoculated subcutaneously in the axilla of nude mice, with a cell inoculation volume of 2*10 6 Human breast cancer cell line MCF-7 in logarithmic growth phase was prepared into 1×10 8 / ml cell suspension, 0.1ml was inoculated into the right axilla of nude mice. The diameter of the transplanted tumor of nude mice was measured with a vernier caliper. When the tumor grew to 100-300mm 3 The animals were then randomly divided into groups. The anti-tumor effect of the test substance was dynamically observed by measuring the tumor diameter. The tumor diameter was measured once every 3 days. The administration volume was 0.5 ml / 20 g. After 28 days, the mice were killed and the tumor was surgically removed and weighed.

[0087] 4) Anatomical experimental design and tumor histopathological examination

[0088] At the end of the experiment, all mice were killed by cervical dislocation. The tumors were removed and weighed to calculate the tumor growth inhibition percentage (TGI%), and then stored in 4% paraformaldehyde. For subsequent tumor tissue staining. TGI% = (W negative control group-W experimental group) / W negative control group × 100% W negative control group is the average tumor weight of mice in the negative control group; W experimental group is the average tumor weight of mice in the drug-treated group. The number of days of death of the remaining mice was recorded for survival studies. The "Guidelines for Tumor Induction in Mice and Rats (Updated in May 2013)" states that "the tumor size of adult mice cannot exceed 20 mm (2.0 cm) in any direction", so any animal with a tumor size exceeding 20 mm is defined as terminated by the study.

[0089] Antitumor pharmacodynamics experiments were conducted when the mouse tumor size reached 62.50±10mm3 (5mm*5mm*5mm). The first and second groups were injected intraperitoneally with original paclitaxel and water-soluble paclitaxel injection (8mg·kg-1 according to the instructions for the treatment of solid tumors), respectively. The third group was used as a negative control group without any treatment. After three weeks, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, and stomach tumors were obtained and measured and weighed. The dissected tissues were histologically fixed and kept for sectioning.

[0090] 5) Results and Discussion

[0091] Test results such as Fig.11 As shown, Fig.11 In the experiment, AC were the control group, the group injected with original paclitaxel, and the group injected with water-soluble paclitaxel. Fig.11 It can be seen that the tumors of the mice in the control group without medication were larger. In comparison, the tumors of the mice in the group injected with the original paclitaxel agent and the group injected with the water-soluble paclitaxel agent were smaller than those in the control group, but the tumors in the group injected with the water-soluble paclitaxel agent were relatively smaller, indicating that both the injection of the original paclitaxel agent and the water-soluble paclitaxel agent can inhibit the growth of tumors, among which the injection of the water-soluble paclitaxel agent has a better inhibitory effect.

[0092] Fig.12 This is the curve of tumor volume change. It can be seen that the tumor volume of mice in the group injected with original paclitaxel and the group injected with water-soluble paclitaxel is smaller than that of the control group, and the tumor in the group injected with water-soluble paclitaxel is relatively smaller, indicating that both the injection of original paclitaxel and the water-soluble paclitaxel can inhibit tumor growth, but the inhibitory effect of the injection of water-soluble paclitaxel is better.

Claims

1. A highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound, the compound having a structural formula as shown in formula (I):

2. The method for synthesizing the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound as claimed in claim 1, characterized in that: The specific steps are as follows: 1) mixing aconitic anhydride and paclitaxel in an anhydrous solvent, and performing an esterification reaction at 90° C. to obtain an esterification reaction product; 2) placing the esterification reaction product obtained in step 1) at 60° C. to evaporate to obtain a viscous product, adding water, stirring, and centrifuging to obtain a supernatant; 3) placing the supernatant obtained in step 2) into a 300 molecular weight dialysis bag and dialyzing it with deionized water for 24 hours, and then freeze-drying it at -20°C to obtain a powdery product; 4) dissolving the powdery product obtained in step 3) in 60° C. water, leaving it to stand at 4° C. and filtering it, and drying the solid to obtain the water-soluble paclitaxel anticancer drug compound.

3. The method according to claim 2, characterized in that In step 1), the molar mass ratio of paclitaxel to aconitic anhydride is 1:

5.

4. The method according to claim 2, characterized in that: In step 1), the anhydrous solvent is pyridine.

5. Use of the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound according to claim 1 in the preparation of anticancer drugs for prevention or treatment.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition is obtained by dissolving the highly water-soluble bisaconitic acid molecule chemically modified paclitaxel compound as claimed in claim 1 in a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutically acceptable carrier or excipient includes water, PBS buffer, physiological saline or glucose solution.

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