CN102125547B - Pharmaceutical composition containing gambogic acid medicament and preparation method thereof - Google Patents

Abstract

The invention discloses a pharmaceutical composition containing gambogic acid drugs and a preparation method thereof. The invention specifically relates to a pharmaceutical composition containing gambogic acid drugs, which is characterized in that it comprises a gambogic acid drug that forms micelles with amphiphilic high polymer conjugates. In the present invention, the gambogic acid drugs are encapsulated in the amphiphilic high polymer conjugate through physical embedding and chemical coupling, which can not only greatly increase the solubility of the gambogic acid drugs, but also significantly improve the storage stability And anti-tumor activity, improve drug safety. Compared with gambogic acid drugs, the medicinal composition has improved curative effect and reduced irritation in treating liver cancer, cervical cancer and other tumors. The present invention also provides a method for preparing the above-mentioned amphiphilic high-polymer conjugate and the pharmaceutical composition containing gambogic acid drugs. The preparation method of the invention is simple, the process is mature, the yield is high, and it is suitable for industrialized production.

Description

A pharmaceutical composition containing gambogic acid drugs and its preparation method

technical field

The invention belongs to the field of pharmaceutical preparations, and relates to a pharmaceutical composition containing gambogic acid drugs and a preparation method of the pharmaceutical composition. The pharmaceutical composition is characterized in that the amphiphilic high-polymer conjugate encapsulates gambogic acid drugs in the form of micelles.

Background technique

Cancer has become a common disease that seriously threatens human life and health around the world. Clinical treatments such as surgery and chemotherapy are effective means to remove tumors, but surgery can only remove tumors that are visible to the naked eye, but cannot remove invisible subclinical lesions and tumor cells that have metastasized or infiltrated into normal tissues; while conventional Due to the fact that the existing anticancer drugs have almost no selectivity for tumor tissues and cells, there are common problems such as low clinical curative effect, high toxicity, difficult to control metastases, and poor drug compliance of patients, which leads to some patients refusing to use drugs or failing to take drugs. situation. Anti-tumor drug treatment of cancer is one of the main methods of clinical treatment at present. However, most of the existing anti-tumor drugs have poor solubility, poor oral absorption, low bioavailability, and fast metabolism in the body. Shorter, in order to improve the curative effect often need to increase the dose or increase the frequency of administration, coupled with the lack of selectivity for normal cells and cancer cells, thus bringing greater toxicity and side effects. Therefore, the development of long-acting, sustained-release and targeted anti-tumor drug preparations is the focus of current research.

The outstanding advantages of anti-tumor targeted preparations are strong pertinence, significant effect, and low side effects; anti-tumor drugs are prepared into liposomes, microcapsules (spheres) and other drug delivery systems to enhance targeting and improve curative effect, and have become tumor target agents. However, as a drug carrier, liposomes still have disadvantages such as low encapsulation efficiency, unsatisfactory target distribution, and poor stability in storage; microcapsules (spheres) have large particle sizes and cannot penetrate The drug is directly delivered to the target tissue through the mucosa or through the systemic circulation, which is not suitable for injection; and liposomes and microcapsules (spheres) are not very effective in the treatment of some solid tumors (such as breast cancer, prostate cancer, colon cancer, etc.) . The polymer conjugate is a very effective way, which connects small molecule hydrophobic drugs to polymer carriers to form a new type of polymer, especially nanoscale polymer particles due to their small size effect and surface, Interface effects, etc., can not only improve drug efficacy, reduce adverse reactions, but also facilitate the use of patients. Its outstanding advantages are: ① increasing the solubility of hydrophobic drugs; ② passive targeting and slow and controlled release; ③ avoiding rapid clearance by the kidneys, prolonging the plasma half-life of drugs, etc.

Garcinia cambogia is extracted from the dry resin secreted by Garciniaceae plants, mainly including gambogic acid, neogambogic acid, allogambogic acid and other components. Gambogic acid is the main active ingredient of Gamboge, which has obvious inhibitory effect on various tumors. The results of the study showed that gambogic acid had higher distribution and longer duration in tumor tissue. The results of research on the molecular level of the mechanism of action of gambogic acid show that: gambogic acid has the characteristics of high efficiency, low toxicity, and multiple targets, and can selectively kill cancer cells without affecting the normal hematopoietic system and white blood cells. It provides an application prospect for finding new anticancer drugs. Gambogic acid is extremely difficult to dissolve in water. Currently reported gambogic acid injections are mainly prepared with boric acid solution, or added with solubilizers such as L-arginine, meglumine, and Tween to prepare gambogic acid freeze-dried preparations. Long-term use of these solubilizers will cause a series of adverse reactions, or require a larger dose to achieve a stable and effective solubilizing effect.

In the previous research, the inventor found that the amphiphilic high-polymer conjugate has a good solubilizing effect, and has a good solubilizing effect on some poorly soluble drugs such as paclitaxel and doxorubicin. The amphiphilic polymeric conjugates used here are based on natural polysaccharides as the backbone, and the carboxyl group-containing hydrophobic drugs are introduced on the carboxyl group of the polysaccharide or the carboxyl group formed by derivatization through a simple alkylene diamine as the linking arm. The conjugate has both a hydrophobic drug part and a hydrophilic polysaccharide part, which greatly enhances the amphiphilicity and has the characteristics of polymer micelles: 1) self-assembly in aqueous solution to form nanomicelles, avoiding the The use of organic solvents, surfactants, cross-linking agents or heating conditions; 2) under the dual action of polysaccharide molecular chains and hydrophobic drugs, the critical micelle concentration of micelles is significantly reduced, the stability time is significantly prolonged, and the drug’s drug loading and encapsulation efficiency; 3) Since the antitumor drug solubilized in the conjugate core is combined with the carrier in a non-covalent manner, the carried drug can be released relatively easily, and the chemically coupled drug The release behaviors complement each other to achieve a programmed drug release effect; 4) The mechanism of action of the anti-tumor drugs that are physically solubilized and chemically coupled can complement each other to enhance the anti-tumor activity and achieve the effect of combined therapy. CN101791411A discloses these techniques.

The use of amphiphilic polymer conjugates to encapsulate gambogic acid drugs has obvious advantages compared with the use of other carriers to encapsulate gambogic acid drugs. For example, gambogic acid, phospholipids, and cholesterol are used in a certain weight ratio to prepare gambogic acid liposomes by thin-film ultrasonic method. But gambogic acid liposome has the following defects: poor physical and chemical stability, and the medicine that is wrapped after storage for a certain period of time leaks easily; Encapsulation efficiency is low, and the highest encapsulation efficiency is only about 80%; Large particle size, lack of vascular permeability, difficult to pass through the intercellular space of liver blood vessels, and easy to be swallowed by the reticuloendothelial system; the preparation process and quality control process of gambogic acid liposomes have high requirements, and it is difficult to achieve large-scale industrialization Production.

In view of the above problems, this patent uses a pharmaceutical composition of gambogic acid drugs and amphiphilic polymer conjugates to construct a new anti-tumor drug delivery system. In this way, gambogic acid is encapsulated in high-polymer conjugates in the form of physical embedding and chemical coupling. The pharmaceutical composition can greatly enhance the pharmacological effect, improve drug safety, and realize drug combination therapy. The efficacy is significantly enhanced, the irritation is significantly reduced, and the ideal drug release effect is achieved.

Contents of the invention

The object of the present invention is to provide a kind of pharmaceutical composition containing gambogic acid medicine for above-mentioned technical problem. The pharmaceutical composition utilizes amphiphilic high-polymer conjugates to coat gambogic acid drugs, and the pharmaceutical composition has good safety, high drug loading capacity, good physiological activity, improved drug efficacy, reduced toxic and side effects, and good stability.

Another object of the present invention is to provide a preparation method of a pharmaceutical composition containing the above gambogic acid-containing drug.

Another object of the present invention is to provide the application of the above-mentioned pharmaceutical composition containing gambogic acid drugs in pharmacy.

In the present invention, the amphiphilic high-polymer conjugate has a solubilizing effect on gambogic acid drugs, and the solubilizing effect is better than paclitaxel, doxorubicin and other poorly soluble antitumor drugs. The conjugates can self-assemble into nano-micelles with anti-tumor activity in aqueous medium, wrap gambogic acid drugs, and effectively solve the solubility problem of gambogic acid.

The gambogic acid drugs described in the pharmaceutical composition containing gambogic acid drugs mainly include total gambogic acid, gambogic acid, neogambogic acid, allogambogic acid and the above-mentioned drugs and basic amino acids, alkali metal ions, A pharmaceutical complex formed by nitrogen-containing organic basic compounds and glucosamine compounds.

The amphiphilic polymer conjugate described in the pharmaceutical composition containing gambogic acid drugs, wherein the selected polysaccharides include polysaccharides originally containing carboxyl groups including unfractionated heparin, low molecular weight heparin, desulfated heparin, hyaluronic acid, Chondroitin, polysulfated chondroitin, alginic acid, and carboxyl-containing polysaccharide derivatives include polysaccharide chitosan, carboxymethyl chitosan, succinyl chitosan, dextran, fungal polysaccharide, carboxymethyl lentinan. The hydrophobic groups are pharmaceutically active or pharmacologically active molecules containing carboxyl groups, including gambogic acid, neogambogic acid, allogambogic acid, all-trans retinoic acid, 9-cis retinoic acid, methotrexate, amino Pterin, raltitrexed, pemetrexed, chlorambucil, aminolevulinic acid, ginsenosides, oleanolic acid, ursolic acid, aspirin, diclofenac, ketoprofen, ibuprofen, fenbu Fen, mefenamic acid, meclofenamic acid, indomethacin, sulindac, acemetacin, benzydamine, etodolac, naproxen, ibuprofen, norfloxacin, Ofloxacin, ciprofloxacin, pipemidic acid, levofloxacin, enoxacin, lomefloxacin, pefloxacin, fleroxacin, tosufloxacin, sparfloxacin, tomefloxacin, Baroxacin, edatrexate, and bile acids including cholic acid, ursodeoxycholic acid, lithocholic acid, and chenodeoxycholic acid.

The preparation method of described amphiphilic high polymer conjugate, comprises the following steps:

Dissolve the carboxyl-containing hydrophobic drug in an appropriate organic solvent, use alkylenediamine as the linking arm, dicyclohexylcarbodiimide (DCC) and hydroxysuccinimide (NHS) as the activator to carry out the condensation reaction, Obtain the active intermediate of free one-terminal amino group; dissolve the polysaccharide containing carboxyl group or derivatized into carboxyl group in the reaction solvent, and pass through 1-ethyl-(-dimethylaminopropyl) carbodiimide to the active intermediate obtained (EDC) is an activator for further carboxyl and amino condensation reactions.

In the preparation method, the appropriate organic solvent is preferably selected from N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

In the preparation method, the linking arm is an alkylene diamine structure with 2 to 12 carbon atoms.

The preparation method, wherein the reaction solvent is preferably selected from water, or formamide, or N, N-dimethylformamide and water, or formamide and water, or N, N-dimethylformamide and formamide Mixed solvents.

The preferred preparation method of the pharmaceutical composition containing gambogic acid drugs in the present invention is as follows: the amphiphilic high-polymer conjugate and water are dissolved in a ratio of 3 to 50:1000 by weight to obtain the conjugate nano-micelle ; After dissolving the therapeutically effective amount of insoluble or slightly water-soluble gambogic acid drugs with a pharmaceutically acceptable solvent, after mixing with the conjugated nanomicelle, after ultrasonic or high-pressure homogeneous treatment, the solution is used Organic solvents and small molecules are removed by dialysis or ultrafiltration or column separation to prepare solution-type preparations.

The pharmaceutically acceptable solvent is preferably one or more mixed solvents of methanol, ethanol, methylene chloride, chloroform, dimethyl sulfoxide, and N,N-dimethylformamide. The pharmaceutically acceptable solvent is further preferably ethanol. The micelle prepared by this method has high drug loading capacity, high encapsulation efficiency, sustainable drug release, and prolongs the time in blood circulation.

Research and development found that, taking the gambogic acid-hyaluronic acid conjugate and the pharmaceutical composition of gambogic acid as an example, the dosage of the gambogic acid-hyaluronic acid conjugate and the gambogic acid are different, and the drug loading is different. When the amount of gambogic acid is too little or too much, the drug loading effect is not ideal. The inventor finally determined through experiments that the mass ratio of the amphiphilic polymer conjugate to gambogic acid is 1:0.01-1:1. It is further preferred that the mass ratio of the amphiphilic polymer conjugate to gambogic acid is 1:0.4-1:0.6. The micelle formed by the gambogic acid and the gambogic acid-hyaluronic acid conjugate prepared by the optimal process has a drug loading capacity of up to 38.7%, an encapsulation rate of 88.2%, and the particle size of the prepared micelles up to the nanometer level. The drug content per unit volume of the gambogic acid micelles in the invention is high, and has stable drug loading and encapsulation efficiency. Compared with the gambogic acid crude drug and the gambogic acid liposome, the pharmaceutical composition of the present invention has better stability than the gambogic acid liposome through a stability comparison test. Gambogic acid raw material drug content obviously drops after a certain period of time, and the gambogic acid liposome also has a leakage phenomenon after a certain period of time, and the micellar particle size of the pharmaceutical composition is basically unchanged in the same period of time. The drug content is stable.

The above pharmaceutical composition containing gambogic acid drugs can also be freeze-dried to make a freeze-dried preparation. A freeze-drying protective agent can be added when making a freeze-dried preparation, and the freeze-drying protective agent can be selected from one or more of dextran, mannitol, glucose, lactose, sucrose, and trehalose. The amount of the lyoprotectant is calculated according to the volume of the micellar solution, preferably 0.01-0.1 part of the lyoprotectant is added to each part of the micelle solution, filtered and then freeze-dried.

The freeze-dried preparation containing gambogic acid drugs prepared by the above method does not contain any organic solvent, and can be reconstituted with water for injection, glucose injection or physiological saline before clinical use. The freeze-dried preparation of the present invention dissolves rapidly when redissolving, and the solution clarify. Gambogic acid micellar freeze-dried powder injection can not only significantly improve the storage stability, but also reduce the unsafety of clinical medication.

The pharmaceutical combination preparation of the present invention can be administered to patients in need of this treatment by oral or parenteral administration, and when used for oral administration, it can be made into conventional solid preparations such as tablets, capsules, powders, granules etc., made into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, oral liquids, etc.; for parenteral administration, it can be made into injection solutions, powder injections, water or oily suspensions, etc. Preferred forms are injectable powders, tablets, coated tablets.

Another object of the present invention is to provide a pharmaceutical composition for treating liver cancer, cervical cancer and other tumors, which has better curative effect than gambogic acid and lower irritation and toxicity. The present invention also provides the use of the above pharmaceutical composition in the preparation of medicines for treating liver cancer, cervical cancer and other tumors.

The present invention finds that the pharmaceutical composition containing gambogic acid has no vascular irritation (infusion of rabbit ear vein, equivalent to 1mg gambogic acid/20ml/kg). The hemolysis test of the present invention is carried out with 2% rabbit erythrocyte suspension, which shows that there is no hemolysis and aggregation of erythrocytes within 2 hours at a dose below 0.5ml. When the pharmaceutical composition is administered through blood vessels, the local stimulating side effect is lower than that of gambogic acid alone. Such as: Rabbit ear vein aseptic operation slow infusion of gambogic acid sodium salt, gambogic acid-containing pharmaceutical compositions such as all-trans retinoic acid-hyaluronic acid and gambogic acid combined preparations, 0.9% chloride Sodium injection, once a day, 3 times in a row. The results show that compared with the control group: the pharmaceutical composition containing gambogic acid has no irritation to the rabbit's auricular vein, and the tissue section checks that the rabbit's auricular vein has normal vascular structure, no endothelial damage, no thrombus formation and other pharmacological effects. Changes: Gambogic acid is irritating to the auricular vein of rabbits, and the tissue section examination shows that the auricular vein is slightly dilated, some endothelial cells are necrotic and fall off, and inflammatory reactions can be seen in the surrounding tissues.

The present invention also finds that the pharmaceutical composition containing gambogic acid medicines increases the solubility of gambogic acid medicines in water, and at the same time, the drug effect is also significantly enhanced. Using tetramethyl azozolium salt colorimetric method, it was found that the pharmaceutical composition formed by all-trans retinoic acid-hyaluronic acid and gambogic acid has a better drug effect on cervical cancer Hela cells and liver cancer HepG2 cells than gambogic acid raw materials Significantly improved, it is a drug with good water solubility and high drug efficacy that meets clinical needs.

Beneficial effects of the present invention:

1. The present invention combines gambogic acid drugs with amphiphilic polymer conjugates, and uses nano-micelles composed of hydrophilic shell and hydrophobic core to encapsulate anti-tumor drugs, which can prolong the internal circulation and reduce the phagocytosis of reticuloendothelial cells , increase tumor targeting and improve safety.

2. In the pharmaceutical composition containing gambogic acid drugs provided by the present invention, the amphiphilic high-polymer conjugate utilizes polysaccharides to graft hydrophobic groups, and can spontaneously form nano-micelles in water, which can not only be used for antitumor drugs Because of the nano-micelle structure composed of hydrophilic shell and hydrophobic core, it can prolong the circulation in vivo, reduce the phagocytosis of reticuloendothelial cells, and increase tumor targeting, so it can be physically encapsulated and chemically coupled with anti-tumor drugs , to achieve the effect of combined treatment of cancer;

3. The pharmaceutical composition containing gambogic acid drugs provided by the present invention can be used for injection, oral administration, external application or mucosal administration. It has high safety, particle size can be controlled in 10-1000nm, good solubility and stability.

4. The pharmaceutical composition containing gambogic acid drugs provided by the present invention improves the defects of the current gambogic acid preparations, is economical and practical, has good anti-tumor effect, no blood vessel irritation, and high drug efficacy.

Detailed ways

Below by embodiment the present invention is further described, but following embodiment does not limit the right scope of this patent.

Example 1: Synthesis of all-trans retinoic acid-hyaluronic acid conjugates

Take 10mmol all-trans retinoic acid, 12mmol dicyclohexylcarbodiimide (DCC), 15mmol hydroxysuccinimide (NHS), dissolve in 30ml N,N-dimethylformamide, protect from light and nitrogen , reacted in ice bath for 30min, and then rose to room temperature for 24h. After the reaction, the precipitate was filtered off, and a large amount of ethyl acetate was added to wash the precipitate. The filtrate was extracted, the ethyl acetate layers were combined, and the solvent was removed by rotary evaporation to obtain the activated intermediate ester of all-trans retinoic acid. Dissolve 1mmol of all-trans retinoic acid activated intermediate ester in 10ml of dichloromethane, slowly drop into 3mmol/ml of ethylenediamine in dichloromethane solution under ice-bath conditions, and monitor the reaction by thin layer chromatography (TLC method) After completion, the reaction liquid is extracted, the organic layers are combined, and the obtained product is separated and purified by silica gel column chromatography, which is the active intermediate of all-trans retinoic acid. Take 26mmol of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, add it to the formamide solution containing 0.1mmol of hyaluronic acid, react at room temperature for 15min, and make the active intermediate containing 26mmol of all-trans retinoic acid A solution of N,N-dimethylformamide was slowly added to the above reaction solution, and reacted at room temperature for 24 hours. After the reaction was completed, acetone was added to precipitate the product, and the precipitate was obtained by suction filtration. Add water to redissolve the precipitate, dialyze in water for 3 days, and freeze-dry to obtain the final product of all-trans retinoic acid-hyaluronic acid conjugate.

Embodiment 2: the synthesis of gambogic acid-hyaluronic acid conjugate

Take 10mmol gambogic acid, 12mmol dicyclohexylcarbodiimide (DCC), and 15mmol hydroxysuccinimide (NHS), dissolve them in 30ml N, N-dimethylformamide, keep away from light, under the protection of nitrogen, and place in an ice bath Reacted for 30min, then rose to room temperature for 24h. After the reaction, the precipitate was filtered off, and a large amount of ethyl acetate was added to wash the precipitate. The filtrate was extracted, the ethyl acetate layers were combined, and the solvent was removed by rotary evaporation to obtain the activated intermediate ester of gambogic acid. Dissolve 1mmol of gambogic acid activated intermediate ester in 10ml of dichloromethane, slowly drop into 3mmol/ml of ethylenediamine in dichloromethane solution under ice-bath conditions, and monitor the reaction until complete by thin-layer chromatography (TLC method) Afterwards, the reaction solution is extracted, the organic layers are combined, and the obtained product, which is the active intermediate of gambogic acid, is separated and purified by silica gel column chromatography. Get 26mmol 1-ethyl-(3-dimethylaminopropyl) carbodiimide, add in the formamide solution containing 0.1mmol hyaluronic acid, react at room temperature for 15min, and N containing 26mmol gambogic acid active intermediate , N-dimethylformamide solution was slowly added to the above reaction solution, and reacted at room temperature for 24h. After the reaction was completed, acetone was added to precipitate the product, and the precipitate was obtained by suction filtration. Add water to redissolve the precipitate, dialyze in water for 3 days, and freeze-dry to obtain the final product gambogic acid-hyaluronic acid conjugate.

Example 3: Preparation of self-assembled nanomicelle composition containing gambogic acid and gambogic acid-hyaluronic acid conjugate

(1) Dialysis

Gambogic acid-hyaluronic acid conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Gambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

(2) Emulsion solvent evaporation method

Gambogic acid-hyaluronic acid conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Gambogic acid 9 mg was dissolved in dichloromethane. Then the two were mixed, the probe was sonicated for 30 minutes, and stirred overnight at room temperature to volatilize the dichloromethane, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 4: Preparation of self-assembled nanomicelle composition containing gambogic acid and gambogic acid-heparin conjugate

Gambogic acid-heparin conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Gambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 5: Preparation of self-assembled nanomicelle composition containing gambogic acid and all-trans retinoic acid-hyaluronic acid conjugate

All-trans retinoic acid-hyaluronic acid conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Gambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 6: Preparation of self-assembled nanomicelle composition containing gambogic acid and methotrexate-chondroitin sulfate conjugate

Methotrexate-chondroitin sulfate conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Gambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 7: Preparation of self-assembled nanomicelle composition containing neogambogic acid and gambogic acid-hyaluronic acid conjugate

Gambogic acid-hyaluronic acid conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Neogambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 8: Preparation of self-assembled nanomicelle composition containing neogambogic acid and aspirin-alginic acid conjugate

Aspirin-alginic acid conjugate 18mg, dissolved in 3ml of water and stirred for 1h. Neogambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 9: Preparation of self-assembled nanomicelle composition containing neogambogic acid and ursodeoxycholic acid-hyaluronic acid conjugate

18mg of ursodeoxycholic acid-hyaluronic acid conjugate was dissolved in 3ml of water and stirred for 1h. Neogambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 10: Preparation of self-assembled nanomicelle composition containing allogambogic acid and ursodeoxycholic acid-hyaluronic acid conjugate

18mg of ursodeoxycholic acid-hyaluronic acid conjugate was dissolved in 3ml of water and stirred for 1h. Allogambogic acid 9 mg was dissolved in ethanol (methanol). Then the two were mixed, after the probe was ultrasonicated for 30 minutes, it was dialyzed overnight in double distilled water, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

Example 11: Determination of Gambogic Acid Content in Self-Assembled Nanomicelles of Pharmaceutical Composition

The content determination was carried out by HPLC (LC-2010C, Shimadzu, Japan) method. The mobile phase was methanol:water=93:7 (v/v), containing 0.1% glacial acetic acid, the chromatographic column was Lichrospher C ₁₈ (150×4.6 μm), and the particle size of the column was 5 μm. The flow rate was 1.0 mL/min, the detection wavelength was 360 nm (SPD-10A, UV detector, Shimadzu, Japan), the column temperature was 30° C., and the injected sample volume was 20 μl. The drug loading amount of the sample was calculated by formula (1). The results are shown in Table 1.

Table 1 Examples 3-6 Self-assembled nanomicelles of amphiphilic polymer conjugates loaded with gambogic acid

Amphiphilic polymer conjugates Preparation process Drug loading (%)

Example 3 Dialysis 38.7

Example 3 Emulsion solvent evaporation method 29.5

Example 4 Dialysis 34.5

Example 5 Dialysis 31.2

Example 6 Dialysis 28.9

Embodiment 12: Determination of content of new gambogic acid in self-assembled nanomicelle of pharmaceutical composition

The content determination was carried out by HPLC (LC-2010C, Shimadzu, Japan) method. The mobile phase was methanol: 1.0 mol/L phosphoric acid=90:10 (v/v), the chromatographic column was Lichrospher C ₁₈ (150×4.6 μm), and the particle size of the column was 5 μm. The flow rate was 1.0 mL/min, the detection wavelength was 360 nm (SPD-10A, UV detector, Shimadzu, Japan), the column temperature was 30° C., and the injected sample volume was 20 μl. The results are shown in Table 2.

Table 2 Example 7 Self-assembled nanomicelles of amphiphilic polymer conjugates loaded with neogambogic acid

Amphiphilic polymer conjugates Preparation process Drug loading (%)

Example 7 Dialysis 34.8

Embodiment 7 Emulsion solvent evaporation method 24.9

Example 13: Investigation on the stability of self-assembled nanomicelles of the pharmaceutical composition of gambogic acid and all-trans retinoic acid-hyaluronic acid conjugate

The particle size, potential and drug content of the drug combination freeze-dried preparations reconstituted in water were used as indicators to investigate the stability of Examples 3-5 in an aqueous environment at 0-4°C; the results are shown in Table 3. The results show that the pharmaceutical composition of gambogic acid drugs and amphiphilic polymer conjugates can exist stably under this condition.

Table 3 Examples 3-5 Stability of self-assembled nanomicelles containing gambogic acid and amphiphilic polymer conjugates

Embodiment 14: The loading situation of the pharmaceutical composition containing gambogic acid drugs

The self-assembled nanomicelle composition containing gambogic acid and all-trans retinoic acid-hyaluronic acid conjugate is prepared according to Example 5, and gambogic acid liposomes are prepared using gambogic acid, phospholipids, and cholesterol According to a certain weight ratio, it is prepared by thin film ultrasonic method. The results are shown in Table 4.

Table 4 The drug-loading situation of the pharmaceutical composition containing gambogic acid drugs

The results show that the drug loading and encapsulation efficiency of the pharmaceutical composition micelles formed by amphiphilic high-polymer conjugates encapsulating gambogic acid are significantly higher than that of gambogic acid liposomes, and the encapsulation efficiency can reach up to 88.2%. And the highest encapsulation efficiency of gambogic acid liposome is only 80.2%. On the 7th day, the encapsulation efficiency of gambogic acid liposomes dropped to 57.1%, and the particle diameter also increased rapidly from 232.1nm to 410.6nm, while the encapsulation efficiency and particle diameter of the polymer micelles containing gambogic acid were basically Keeping unchanged, it shows that the stability of the polymer micelles containing gambogic acid is obviously better than that of gambogic acid liposomes.

Embodiment 15: Laser confocal microscope (cLsM) observes the cell uptake experiment of the pharmaceutical composition containing gambogic acid medicine

1. Preparation process

Dissolve 9 mg of all-trans retinoic acid-hyaluronic acid conjugate in 3 ml of water and stir for 1 hour. Coumarin 1.5ug was dissolved in dichloromethane. After the two were mixed, the probe was sonicated for 30 minutes, stirred overnight at room temperature to evaporate dichloromethane, centrifuged (3000 rpm) for 15 minutes, filtered with a 0.45 μm filter membrane, and freeze-dried.

2. Observation method

Pre-incubated breast cancer B21 cells were made into cell suspension, and added to 24-well plates at a cell concentration of 1×10 ⁵ /ml, 300 μl per well, set up five duplicate wells, and cultured in a 37°C 5% CO ₂ incubator 24h. Remove the cell suspension, add 10ul coumarin-loaded high-polymer conjugate micelles and 300ul serum-free DMEM medium to each well, and incubate in a 5% _CO2 incubator at 37°C for 0.5, 1, 2, and 4 hours Finally, remove the upper liquid, wash three times with PBS, remove after curing with 4% paraformaldehyde for 20 min, wash three times with PBS, stain with Hoechs for 15 min at room temperature and avoid light, then wash three times with PBS, shaking each time Shake for 10 minutes and remove; after mounting, observe and take pictures under a laser converging microscope.

According to the experimental results, the coumarin-loaded high-polymer conjugate micelles had entered the nucleus at 1 hour, and the fluorescence intensity in the nucleus was significantly higher than that in the cytoplasm, and the fluorescence intensity in the nucleus increased with time. It shows that the drug-loaded micelles have nuclear targeting and can successfully enter the nucleus, and this cellular uptake is time-dependent.

Embodiment 16: the in vitro antitumor effect of the pharmaceutical composition containing gambogic acid drugs

HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin, washed and centrifuged, and then prepared into a cell suspension, and 180 μl of the cell suspension (0.5× ¹⁰ cells/ml) was inoculated in a 96-well plate In this method, the cell plate was placed in a 37°C incubator and incubated for 24 hours under 5% CO ₂ , and the cell adherent growth was observed under a microscope. Add 20 μl of different concentrations (5, 10, 20, 30, 40, 50, 100 μg/ml) of gambogic acid solution, corresponding to the same concentration of gambogic acid-containing polymer micelle solution (prepared in Example 5), two Affinity polymer conjugate carrier solution (1000 μg/ml), blank control group was added an equal volume of culture solution. After 24 hours, add 31.5 μl of 5 mg/ml MTT solution to each well, continue to cultivate for 4 hours, absorb the original culture solution, add 200 μl DMSO to each well to dissolve the crystals, measure the OD value of each well at 570 nm with a microplate reader, and calculate The cell growth inhibition rate at different concentrations was calculated by IC ₅₀ . The above experiments were repeated 5 times for each group, and 3 replicate wells were set for each concentration. The results are shown in Table 5.

Cell migration inhibition rate=(1-experimental OD value/blank control group OD average value)×100%

Table 5 The inhibitory effect of each group of drugs on liver cancer HepG2 cells

Drug _IC50 (μg/mL)

Polymeric micelles containing gambogic acid 1.65

Gambogic acid API 3.57

The results showed that the polymer micelles containing gambogic acid and the gambogic acid raw materials had obvious growth inhibitory effect on human liver cancer HepG2 cells in the concentration range of 0.5-10.0 μg/mL, and with the increase of drug concentration, the The growth inhibitory effect of HepG2 cells was also enhanced, showing a significant dose-dependence. The study also found that the IC ₅₀ of the gambogic acid-containing polymer micelles was smaller than that of the gambogic acid raw materials, indicating that the cytotoxicity of the gambogic acid-containing polymer micelles was higher than that of the gambogic acid raw materials. Because the screening of conventional anti-tumor compounds is reflected by the cytotoxicity of the compounds, the pharmaceutical composition prepared from gambogic acid in the form of drug-loaded micelles has better anti-tumor activity and significantly improved drug efficacy.

Example 17: Observation of cell morphology by HE staining

Take the HepG2 cells in the logarithmic growth phase, digest them with 0.25% trypsin, adjust the cell density to 1× ¹⁰⁵ cells/mL, seed them on clean coverslips in a 6-well plate, and divide them into the following 4 groups: (1) Blank control Group (2) blank micelle group: 100 μg/mL amphiphilic high polymer conjugate (3) drug-loaded micelle group: 2 μg/mL and 10 μg/mL gambogic acid polymer micelles (prepared in Example 5 ) (4) API group: Gambogic acid at 2 μg/mL.

After 48 hours of action, carry out HE staining, suck off the culture medium, wash the coverslip with PBS solution, fix the cells at room temperature for more than 30 minutes, wash with PBS for 2×1 minute, stain with hematoxylin solution for 5-10 minutes, soak in tap water, dilute hydrochloric acid Dip in alcohol solution for several seconds, tap water, nuclei blue in light ammonia water for 3-5 minutes, tap water, Yin Hong dyeing solution for 5-10 min, tap water, 70%, 80%, 95% gradient alcohol dehydration, The xylene was transparent, and the slides were sealed with neutral gum, and the cell morphology of each group was observed under a microscope.

The results showed that: Microscopic observation found that HepG2 cells in the control group grew adherently, with fewer granules, obvious outlines of nucleoli and nuclear membranes, and adjacent cells grew and fused into sheets; After 24 hours, the HepG2 cells treated with the polymer micelle pharmaceutical composition found that the cells gradually fell off from the wall, the number of cells decreased, and the refractive properties deteriorated, indicating that the growth and proliferation of the cells were obviously inhibited; after 10 μg/mL of gamboge The HepG2 cells treated with the acid polymer micelle pharmaceutical composition and the gambogic acid raw material drug, this phenomenon is more obvious, the cells are almost all shed, the number is greatly reduced, and the cell growth is extremely inhibited.

Claims (6)

1. A pharmaceutical composition containing gambogic acid drugs, characterized in that it contains gambogic acid drugs and amphiphilic high-polymer conjugates, and the amphiphilic high-polymer conjugates wrap gambogic acid in the form of micelles Drugs; the gambogic acid drugs are selected from total gambogic acid, gambogic acid, new gambogic acid, allo-gambogic acid and the drugs formed by the above-mentioned drugs and basic amino acids, alkali metal ions, nitrogen-containing organic basic compounds Complex; the amphiphilic high-polymer conjugate, characterized in that the polysaccharide is originally carboxyl-containing hyaluronic acid, heparin, chondroitin sulfate, alginic acid, and the hydrophobic group is carboxyl-containing pharmaceutical activity or Pharmacologically active molecules gambogic acid, all-trans retinoic acid, methotrexate, aspirin, ursodeoxycholic acid; the weight ratio of the amphiphilic polymer conjugates to gambogic acid drugs is 1:0.4- 1:0.6. 2. The preparation method of the pharmaceutical composition according to claim 1, comprising the steps of: dissolving the amphiphilic high-polymer conjugate and water in a ratio of 3-50: 1000 by weight to obtain the conjugate nanomicelle Dissolving the therapeutically effective amount of gambogic acid drugs in a pharmaceutically acceptable solvent, mixing with the conjugated nano-micelle, homogenizing with ultrasound or high pressure, and then evaporating the solution, dialysis or ultrafiltration Or column separation to remove organic solvents and small molecules to make solution-type preparations, or absorb the solution on the surface of solid excipients to prepare solid preparations or lyophilize to prepare freeze-dried preparations. 3. according to the preparation method of the amphiphilic high polymer conjugate in the described pharmaceutical composition of claim 2, it is characterized in that comprising the following steps: the hydrophobic medicine containing carboxyl group is dissolved in suitable organic solvent, adopts alkylene Diamine is used as the connecting arm, dicyclohexylcarbodiimide and hydroxysuccinimide are used as activators to carry out condensation reaction to obtain an active intermediate with free one-terminal amino group; dissolve carboxyl-containing or derivatized carboxyl-containing polysaccharides in the reaction solvent In, with the active intermediate obtained, 1-ethyl-(3-dimethylaminopropyl) carbodiimide is used as an activator, and further carboxyl and amino condensation reactions; the resulting high-polymer conjugates have good amphiphilic It can spontaneously form nanomicelles in aqueous medium. 4. The pharmaceutical composition according to claim 1, characterized in that the composition can be used for injection, oral or external administration. 5. the application of pharmaceutical composition according to claim 4 in pharmacy, it is characterized in that described injection administration is selected from injection, lyophilized powder injection, and oral administration is selected from tablet, capsule, pill, syrup , granule, oral solution, external administration is selected from patch, liniment, lotion, gel, paint, ointment. 6. The pharmaceutical composition according to claim 1, its application in the preparation of medicines for the treatment of liver cancer, cervical cancer and other tumors.