WO2008080369A1

WO2008080369A1 - Steady liposomal composition

Info

Publication number: WO2008080369A1
Application number: PCT/CN2008/000014
Authority: WO
Inventors: Shankui Liu; Limin Li; Jianhui Guo
Original assignee: Shanghai Allist Pharmaceuticals, Inc.
Priority date: 2006-12-30
Filing date: 2008-01-02
Publication date: 2008-07-10
Also published as: CN100496609C; CN101002733A

Abstract

A steady liposomal composition is manufactured by using hydrogenated saturated phospholipid, cholesterol, and sodium or potassium salt of cholesterol sulfate as main film-forming lipid, using sucrose as lyophilizing excipient, and using the method of thin film evaporation-lyophilizing. This liposome is suitable for carrier of anticancer drug paclitaxel.

Description

A stable liposome composition

The invention belongs to the field of pharmaceutical preparations, and in particular relates to a stable liposome and a composition thereof, and a use and preparation method of the liposome composition for encapsulating paclitaxel, and the use of the paclitaxel liposome composition. technical background

The anticancer drug paclitaxe is a natural product extracted from the bark of Taxus brevifolia Nutt.: for the treatment of ovarian cancer, breast cancer and non-small cell lung cancer. However, because of its extremely low solubility in water, it is soluble only in organic solvents such as methanol, ethanol, and dimethyl sulfoxide, which makes it extremely difficult to intravenously administer paclitaxel. One of the well-known methods for solving its solubility is to dissolve paclitaxel using the surfactant polyoxyethylene castor oil and ethanol as a solvent, such as the 1992 Food and Drug Administration (FDA) approved paclitaxel injection (US Bristol-Myers Squibb) The company produces, the trade name is Taxol, and the trade name registered in China is Taisu. Although polyoxyethylene castor oil can increase the water solubility of paclitaxel, it can release histamine due to its degradation in the body, which may cause a severe oversensitivity reaction. In addition, paclitaxel itself has hematological toxicity, myelosuppression, leukopenia, thrombocytopenia, anemia, and other toxicities.

Therefore, it is desired to prepare a stable preparation capable of reducing the side effects of paclitaxel, on the one hand, avoiding allergic reactions caused by polyoxyethylene castor oil and ethanol complex solvent, and on the other hand reducing the systemic toxicity of paclitaxel.

The use of liposomes as carriers for the administration of paclitaxel is one way to achieve this. The commonly used film-forming materials for liposomes include amphiphilic substances such as phospholipids, cholesterols, and sphingomyelins. Phospholipids are usually used as the main constituents of lipid bilayer membranes, and cholesterol regulates fluidity and flexibility of membranes. play an important role.

Basic studies of paclitaxel liposomes that began in the early 1990s have shown that paclitaxel liposomal formulations have similar efficacy compared to injections, but have reduced toxicity and improved tolerance. There are a number of published technical solutions in the field of paclitaxel liposome research, such as Sharma-A, in Int-J-Cancer 1997, 71, 103-107, which reports paclitaxel liposomes made from three synthetic phospholipids. , the cationic liposome mentioned in CN1378443A, the paclitaxel nanomagnetic targeting preparation prepared by using ultrafine magnetic fluid mentioned in CN1399958A, the charged paclitaxel prepared by phosphatidylglycerol (PG) mentioned in WO9513053 Liposomes prepared by the special phospholipid cardiolipids mentioned in plastids, W09318751, US5424073, US5648090, CN 1275076A, CN1714094A, amino acid-containing paclitaxel liposomes mentioned in CN1291474A, mentioned in CN1391891A Paclitaxel stealth liposomes prepared by amphiphilic polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) modified liposome membrane. Injectable paclitaxel liposomes that have been marketed in China are produced by Nanjing Cisco Pharmaceutical Co., Ltd.

H20030357, the product name is Lipus, according to its instructions, there is still a risk of hypersensitivity reaction, and it is necessary to pre-inject dexamethasone for anti-allergic pretreatment, compared with paclitaxel injection, in terms of tolerance. There is no significant improvement.

In the above-mentioned technical solutions, the use of saturated phospholipids is not clearly defined. As is well known to those skilled in the art, commonly used lipid materials such as natural lecithin and natural soybean phospholipids contain unsaturated fatty acid groups which are easily oxidatively hydrolyzed into peroxides, malondialdehydes, fatty acids and lysophospholipids. Further hydrolyzed to a glycerophosphoric acid complex, a fatty acid or the like. Oxidative hydrolysis of phospholipids also reduces membrane fluidity and promotes drug leakage. These unstable factors are not conducive to the preparation, storage and clinical safe use of liposome products. In addition, the compatibility or compatibility of charged liposomes with serum or blood, cells, tissues is often unpredictable, as large aggregates of strong cation electrical properties in the blood cause the formation of thrombus in the body. Meanwhile, as is well known to those skilled in the art, the charge can predict the stability of the bimolecular membrane, and the liposome formed by the charged phospholipid increases the repulsive force between the liposome vesicles, making it difficult to coagulate and precipitate, but at the same time Phospholipids with the same charge in the bimolecular membrane can cause instability of the membrane structure, so excessively high charge is not always beneficial for liposome-stabilized encapsulating drugs. In addition, the invisible liposome can stabilize the liposome; it can effectively prolong the circulation time of the liposome in vivo, but it adopts the amphiphilic polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) price. expensive. In addition, cardiolipid, a special lipid material, also suffers from overpriced.

In liposomes, the binding of cholesterol to the bilayer membrane formed by phospholipids achieves membrane stability and proper fluidity and flexibility. The cholesterol content has a significant effect on the drug coverage and particle size. Derivatives of cholesterol are also often used as preparations for liposomes. Cholesterol hemisuccinate as mentioned in U.S. Patent 4,891,208 is used in combination with phosphatidylethanolamine to prepare pH sensitive liposomes. This liposome has a completely negative charge and is inefficiently absorbed by the cells. The sterol skeleton-based amphoteric compound disclosed in CN1492876A, wherein the 3-position of the ring is substituted by one or more amphoteric groups having an isoelectric point between 4 and 9, according to the technical solution disclosed in the patent, complex synthesis is required. Process to produce this cholesterol derivative. Various factors affect the pH in the body, but as recognized by those skilled in the art, controlling the release of the drug based on the pH in the body is not always reliable.

Therefore, if a stable liposome can be prepared from a wide range of common lipid materials while controlling its charge to make its in vivo process more predictable, it will be expected to make up for the shortcomings of the above-mentioned technical solutions and provide a new type. A pharmaceutical carrier of stable quality suitable for industrialization. Summary of the invention The present invention provides a liposome and a composition thereof, which do not use a special charged phospholipid, and also avoid the use of expensive polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE). And cardiolipid (cardiolipid), using common hydrogenated saturated phospholipids, cholesterol, cholesterol sodium sulfate or potassium salt as the main carrier, encapsulating the active ingredient, the liposome composition obtained by the present invention has low charge and uniformity of particle size Preferably, the liposome composition composed of a suitable carrier is very stable in quality and has industrialization prospects, which makes up for the deficiency of the existing liposome technology.

The present invention also provides a method of preparing a liposome composition which is obtained by a thin film evaporation-freeze method to obtain a stable liposome composition.

The present invention also provides a method for simultaneously improving liposome stability and drug encapsulation stability, that is, adding a sodium or potassium salt of a trace cholesterol sulfate to a near-neutral film-forming lipid material to make a lipid The zeta potential of the bulk dispersion is maintained in the range of -15 to -35 millivolts (mV).

In particular, the present invention provides a stable paclitaxel liposome composition, a process for its preparation, and its use in the preparation of an antitumor drug.

The invention also provides a method of treating cancer in a human.

The present invention achieves the above objects by optimizing liposome composition. One of the essential components for preparing the liposome of the present invention is a hydrogenated saturated phospholipid. Usually, the saturated phospholipid contains a saturated fatty acid group having a carbon number of 14 to 18. When the number of carbon atoms is less than 14, the ability of the liposome to accommodate the internal aqueous phase is lowered, and after administration, the stability of the liposome in the blood is lowered. On the other hand, too many carbon atoms, reduced biocompatibility, and due to the increase in carbon chain, the phase transition temperature increases, and a very high temperature is required in the production of liposomes, which is stable to drugs and liposomes. Sex is a negative factor. Accordingly, the saturated phospholipids used in the present invention are selected, for example, from hydrogenated saturated soybean phospholipids or lecithin, dipalmitoylphosphonate choline, distearoylphosphatidylcholine. The saturated phospholipid used in the present invention is not limited to the above phospholipid species, and any phospholipid having a saturated fatty acid group having 14 to 18 carbon atoms is suitable for the purpose of the present invention.

Preferred saturated phospholipids of the invention are selected from the group consisting of hydrogenated saturated soybean phospholipids, hydrogenated saturated lecithin, dipalmitoyl choline choline, distearoylphosphatidylcholine, more preferably hydrogenated saturated soybean phospholipids, hydrogenated saturated lecithins. . The hydrogenated saturated soybean phospholipid preferably has a purity of 97% or more, and its main component is phosphatidylcholine, and contains a trace amount of phosphatidylethanolamine and inositol phospholipid, which is hydrolyzed by the fatty chain to prevent hydrolysis and oxidation.

Another essential component in the preparation of the liposomes of the invention is cholesterol. Cholesterol, together with saturated phospholipids, acts as a film-forming lipid that regulates the fluidity of the lipid bilayer membrane.

The third essential component for preparing the liposomes of the present invention is a derivative of the third position of a class of cholesterol, preferably a sodium or potassium salt of cholesterol sulfate. The cholesterol sulfate sodium salt is an amphiphilic substance, and the US-listed amphotericin colloidal dispersion (trade name AMPHOCIL) uses it as a lipid material to form a plate-like dispersion with the drug. in Such compositions are described in US Pat. No. 4,822,777, US Pat. In the present invention, the sodium salt or potassium salt of cholesterol sulfate, as shown in the formula I and the molecular structure of the formula II, is artificially added to the above-mentioned lipid material in an artificial manner, and an unexpected effect is obtained and stabilized. The average particle size is from 100 to 300 nm, and further concentrates on liposomes from 150 to 250 nm.

Formula I Cholesterol Sulfate Sodium II Cholesterol Sulfate Potassium

The sodium or potassium salt of cholesterol sulfate is a negatively charged amphiphilic substance, which functions as: (1) is added to the film-forming lipid in a trace amount, and does not affect the formation of the film due to the repulsion between charges; (2) Due to the affinity between the hydrophilic end and the hydrophilic end of the phospholipid, the bilayer membrane of the liposome is more stable, and the flexibility of the membrane is increased, and the particle size is concentrated at 100-300 nm, and further concentrated at 150. Liposomes in the range of ~250nm, at the same time, reduce drug leakage; (3) This cholesterol derivative can be safely degraded in the body, there is no risk of use.

One technical solution for realizing the liposome of the present invention is as follows:

The film-forming lipid of the liposome bilayer consists of the following ratios: The molar ratio of hydrogenated saturated phospholipid, cholesterol, cholesterol sulfate sodium or potassium salt is: 100:17~50:0.04~5, preferably 100 :17~50:0.2~5, more preferably, the molar ratio of the three substances is: 100:35~45:0.5~2 _; the paclitaxel is used as the active ingredient, and the molar ratio of the mixture to the hydrogenated saturated phospholipid is: 2~8.5: 100, more preferably 4 to 6:100. In this embodiment, a natural or synthetic vitamin E may further be included as an antioxidant, and the weight ratio to the hydrogenated saturated phospholipid is from 0.13 to 1.25:100, more preferably from 0.3 to 1:100. The above substances are formulated in proportion, and the liposome of the present invention can be obtained by a conventional liposome preparation method, for example, a high pressure homogenization method, a micropore extrusion method, an ultrasonic dispersion method, preferably a high pressure homogenization method. The liposome prepared by the above method has an average particle diameter in the range of 100 to 300 nm, preferably 150 to 250 nm. Methods for the preparation of liposomes in the references mentioned above are incorporated herein by reference.

As will be readily understood by those skilled in the art, the liposomes of the present invention are useful not only as carriers for paclitaxel, but also for other taxanes. Similarly, other anticancer drugs, such as hydroxycamptothecin, can also be used as a delivery vehicle for the liposomes of the present invention. Other drugs, regardless of their water solubility, are acceptable if their price/performance ratio is acceptable. Liposomes of the invention can likewise be employed as carriers.

The above-mentioned liposome containing the active compound can be added to a suitable carrier to prepare a suitable liposome composition for therapeutic use in a suitable formulation. A preferred composition scheme is: adding a lyoprotectant, lyophilizing to obtain a stable composition, and filling it into a suitable container to obtain a preparation for therapeutic use. The lyoprotectant has a stabilizing effect on the integrity of the liposomes during storage and use. Known lyoprotectants are polyols such as glycerin, glucose, sucrose, lactose, trehalose, amino acids and the like. In the present invention, sucrose, lactose, maltose, and glucose are preferred as the lyophilized excipient, and sucrose is more preferred. According to the present invention, the lyoprotectant is 1.5 to 10 times, preferably 4 to 9 times the weight of the hydrogenated saturated phospholipid.

The drug-loaded liposome composition form of the present invention can be prepared by the above freeze-drying method. For example, the active ingredient, the lipid substance, if necessary, and the antioxidant are dissolved in a suitable solvent in a suitable solvent to form a clear solution, at a constant temperature of 50 ° C to 65 ° C, according to a conventional method, such as a reduced pressure evaporation method, If necessary, evaporate under reduced pressure in the presence of an inert gas, remove the solvent from the liquid mixture, form a film, and add an aqueous solution in which the proportioned lyophilized excipient is dissolved, hydrated, sonicated or homogenized to fine The degree is between 0.1 and 0.30 μm, and the bacteria are removed by filtration through a 0.22 μm microporous membrane, and then placed in a suitable container such as a vial, and the solvent is removed from the mixture by freeze drying to obtain a dried drug-containing liposome. Then, the packaging container is sealed or glanded, and an inert gas can be passed before the sealing and glanding.

In the above preparation method of the liposome composition, the amount of the lipid-related solvent to be used is not limited, and the amount of the liquid in which the liposome is dissolved is suitable. Generally, suitable solvents are non-polar or weakly polar and can leave no toxic residues after evaporation. According to a conventional method, for example, in the case of an organic solvent which is usually used, it is selected from methanol, ethanol, chloroform or a mixture thereof. In the present invention, ethanol is preferred. The inert gas used in the present invention is selected from the group consisting of nitrogen, helium, and argon. The dried drug-containing liposome film prepared by the preparation method can be stably stored for a long period of time.

In the present invention, a preferred embodiment is: The active ingredient is docetaxel and its derivatives, more preferably paclitaxel. The paclitaxel injection preparation preparation prepared by the invention does not contain polyoxyethylated castor oil, and avoids the hypersensitivity reaction caused by the polyoxyethylated castor oil, and improves the tolerance compared with the conventional paclitaxel injection. Sex.

The paclitaxel liposomes obtained by the present invention have beneficial effects:

(1) Stability of semi-finished products and preparations

The paclitaxel liposome composition prepared by the present invention was measured for zeta potential between -35 mV and -15 mV before lyophilization, and was allowed to stand at room temperature for 24 hours, during which time the zeta potential was measured and remained substantially unchanged. The liposomes remain suspended in the suspended composition without aggregation. After reconstitution, it was measured in the same manner for 24 hours, and liposome aggregation did not occur. The particle size and the measurement results after reconstitution indicate that the particle size distribution is from 100 to 250 nm. And the same Liposomes that were formulated but did not contain cholesterol sulfate settled after standing for two hours.

(2) The effectiveness and safety of treating cancer

In the animal test, the mice in the paclitaxel liposome administration group showed no toxic side effects, and the physiological indexes were normal, while the animals in the same dose of the Taxol group showed transient coma after administration. The preliminary anti-tumor test results showed that there was no significant difference in the tumor inhibition rate between liposome and other doses of Taxol. From this, it can be seen that the paclitaxel liposome of the present invention improves the tolerance of the organism in the case where the tumor inhibition rate is comparable. The liposome composition prepared by using paclitaxel as an active ingredient avoids the hypersensitivity reaction caused by the paclitaxel preparation containing polyoxyethylated castor oil. According to the particle size distribution of paclitaxel liposome after reconstitution and the results of preliminary acute toxicity test, it can be analyzed that it has passive targeting characteristics after intravenous injection, and is distributed in the reticuloendothelial system, thereby reducing the systemic toxicity of paclitaxel and improving the systemic toxicity of paclitaxel. Patient medication tolerance.

(3) Possibility of industrialization

The liposome compositions of the present invention employ saturated phospholipids and cholesterol as film-forming lipids, all of which are commercially available pharmaceutical preparation materials. Avoid the use of expensive materials such as phospholipid-cardiolipin, reduce costs, and have more industrial prospects. The preparation process of the invention is simple, the steps are few, and the preparation equipment used is a conventional equipment, which is convenient for industrial production.

The paclitaxel liposome composition obtained by the invention can be safely used in mammals in an appropriate manner and in a reasonable dose, and can be used as a therapeutic agent for anti-tumor in mammals, especially for human ovarian cancer, ovarian metastatic cancer, breast cancer, The treatment of non-small cell lung cancer can also be used in combination with cisplatin for the treatment of cancer.

The method for treating paclitaxel liposomes of the present invention for treating human cancer comprises administering to a cancer patient a paclitaxel liposome or liposome composition comprising an effective dose of paclitaxel, the cancer species comprising ovarian cancer, ovarian metastatic cancer, breast cancer and Non-small cell lung cancer. Methods for administration to cancer patients include intravenous or intraperitoneal injections or intratumoral injections.

The following is a special description of the terms used in this article:

The term "liposome" as used herein, as is known to those skilled in the art, refers to a closed vesicle which is polymerized primarily from amphiphilic phospholipid molecules and cholesterol. In this context, it refers to a single compartment liposome in the general sense.

The term "liposome composition" as used herein refers to a formulation composition suitable for clinical use consisting of a liposome and a suitable carrier.

The term "film-forming lipid" as used herein, refers to a material that constitutes a bilayer of a liposome, herein referred to as a population of phospholipids and cholesterol and cholesterol derivatives, herein "bilayer material" and "film-forming lipid" Have the same meaning.

The term "saturated phospholipid" as used herein, means that the fatty acid groups in the phospholipid molecule are substantially saturated, i.e., the fatty acid group is substantially free of "C=C" double bonds. The terms "hydrogenated phospholipids" and "hydrogenated saturated phospholipids" have the same meaning as the term. The term "particle size" as used herein refers to the average particle size of liposomes as measured by a laser particle size analyzer.

The term "cholesteryl sulfate sodium or potassium salt" as used herein refers to a compound of the formula I and formula II. DRAWINGS

Fig. 1 is an electron micrograph of the liposome obtained in Example 1.

Figure 2 is the particle size of the liposomes prepared in Example 1 before lyophilization.

Fig. 3 is a particle size distribution after reconstitution of the liposome obtained in Example 1. detailed description

The present invention will now be described in further detail with reference to the following examples. In the examples, the weight of the material obtained by conversion of the molar ratio is charged. Since the phospholipids are mostly a mixture, the approximate molecular weight is taken in the calculation. The molecular quantities of the substances involved are as follows:

Paclitaxel 853.9

Hydrogenated saturated soybean phospholipid 762

Hydrogenated saturated lecithin 780

Distearoylphosphatidylcholine 760

Dipalmitoylphosphatidylcholine 740

Cholesterol 386.6

Sodium cholesterol sulfate 489

Cholesterol sulfate potassium 523

The examples are given for illustrative purposes and are not intended to limit the scope of the invention.

Example 1 Preparation of paclitaxel liposome

Prescription:

Paclitaxel 0.37g

Hydrogenated soybean phospholipid 4g

Cholesterol lg

Sodium cholesterol sulfate 0.05g

Vitamin E 0.055g

Sucrose 15g Water for injection 120ml

Weigh the prescribed amount of paclitaxel, hydrogenated soybean phospholipid, cholesterol, sodium cholesterol sulfate, vitamin E into 100ml of absolute ethanol, dissolve it, and then use a rotary evaporator to remove the solvent in the presence of nitrogen to prepare a film. Place in a desiccator overnight to remove residual solvent. The sucrose solution dissolved in water for injection was added, stirred in a water bath at 55 ° C for 1 hour, ultrasonically dispersed, homogenized by a high-pressure homogenizer, and 0.22 μm PVDF membrane was aseptically filtered, and placed in a vial, and freeze-dried. The vial was purged with nitrogen and the gland was sealed.

According to the method of the Chinese Pharmacopoeia 2005 edition two appendix XI X E, the encapsulation efficiency of the sample was 93.0%.

According to the Chinese Pharmacopoeia 2005 edition two appendix XI X E method, the liposome sample before freeze-drying was determined by laser particle size analyzer, and the average particle size was 138.6 nm, and the average particle size of the reconstituted sample for injection water was 178.5 nm. The electron micrograph is shown in Figure 1. The particle size distribution is shown in Figure 2 and Figure 3.

Example 2 Preparation of paclitaxel liposome

Prescription:

Paclitaxel 0.09g

Hydrogenated soybean phospholipid 4g

Cholesterol 0.4g

Cholesterol Sulfate Potassium 0.006g

Vitamin E 0.012g

Lactose 10g

Water for injection 150ml

The prescribed amount of paclitaxel, hydrogenated soybean phospholipid, cholesterol, potassium cholesteryl sulfate, and vitamin E were weighed and added to 100 ml of absolute ethanol. After dissolution, a film was prepared by a rotary evaporator, placed in a vacuum desiccator overnight, and the residual solvent was removed. Add sucrose solution dissolved in water for injection, water bath at 58 ° C, magnetic stirring for 1 hour, ultrasonic dispersion, using 0.8, 0.4, 0.2 micron polycarbonate film in sequence, high pressure extrusion with extruder, into a vial, frozen Dry, gland sealed.

The average particle diameter of the sample before lyophilization was 164.6 nm and the encapsulation efficiency was 92.1% as measured in the same manner as in Example 1. Example 3 Preparation of paclitaxel liposome

Prescription:

Paclitaxel 0.18g

Hydrogenated soybean phospholipid 4g Cholesterol 0.9g

Sodium cholesterol sulfate 0.013g

Vitamin E 0.04g

Sucrose 25g

Water for injection 150ml

The prescribed amount of paclitaxel, hydrogenated soybean phospholipid, cholesterol, sodium cholesterol sulfate, and vitamin E were weighed and added to 100 ml of absolute ethanol. After dissolution, a film was prepared by a rotary evaporator, placed in a vacuum desiccator overnight, and the residual solvent was removed. Add sucrose solution dissolved in water for injection, 60 ° C water bath, magnetic stirring for 1 hour, ultrasonic dispersion, homogenization with high pressure homogenizer, extrusion with 0.2 micron polycarbonate film, 0.22 micron PVDF membrane sterile filtration, dispensing Into the vial, freeze-dried, gland sealed.

The average particle diameter of the sample before lyophilization was 152.6 nm and the encapsulation efficiency was 90.6% as measured in the same manner as in Example 1. Example 4 Preparation of paclitaxel liposomes

Prescription

Paclitaxel 0.2g

Hydrogenated soybean phospholipid 4g

Cholesterol 0.7g

Potassium Chromate Sulfate 0.012g

Vitamin E 0.04g

Sucrose 15g

Water for injection 150ml

The prescribed amount of paclitaxel, hydrogenated soybean phospholipid, cholesterol, potassium cholesteryl sulfate, and vitamin E were weighed and added to 100 ml of absolute ethanol. After dissolution, a film was prepared by a rotary evaporator, placed in a vacuum desiccator overnight, and the residual solvent was removed. Add sucrose solution dissolved in water for injection, water bath at 50 ° C, magnetic stirring for 1 hour, ultrasonic dispersion, homogenization by rolling homogenizer, extrusion with 0.2 micron polycarbonate membrane, sterile filtration of 0.22 micron PVDF membrane, Fill the vial, freeze dry, and seal the lid.

The average particle diameter of the sample before lyophilization was 163.8 nm and the encapsulation efficiency was 93.2% as measured in the same manner as in Example 1. Example 5 Preparation of paclitaxel liposome

Prescription

Paclitaxel 0.15g Dipalmitoylphosphatidylcholine 2g

Cholesterol 0.5g

Sodium Cholesterol Sulfate 0.005g

Vitamin E 0.025g

Sucrose 6.5g '

3⁄4 water for injection 75ml

The prescribed amount of paclitaxel, dipalmitoylphosphatidylcholine, cholesterol, sodium cholesteryl sulfate, and vitamin E were added to 100 ml of absolute ethanol, dissolved, and then formed into a film by a rotary evaporator, placed in a vacuum desiccator overnight. Remove residual solvent. The sucrose solution dissolved in water for injection was added, stirred in a water bath at 58 ° C for 1 hour, ultrasonically dispersed, sequentially subjected to a 0.8, 0.4, 0.2 micron polycarbonate film, extruded by a high pressure extruder, and placed in a vial, frozen. Dry, gland sealed.

The average particle diameter of the sample before lyophilization was 168.6 nm and the encapsulation efficiency was 96.4% as measured in the same manner as in Example 1. Example 6 Preparation of paclitaxel liposome

Prescription

Paclitaxel 0.15g

Hydrogenated lecithin 4g

Cholesterol 0.4g

Cholesterol sulfate potassium O.OOlg

Sucrose 35g

Water for injection 200ml

The prescribed amount of paclitaxel, hydrogenated lecithin, cholesterol, and potassium cholesteryl sulfate was added to 100 ml of anhydrous ethanol, dissolved, and then formed into a film by a rotary evaporator, placed in a vacuum desiccator overnight to remove residual solvent. Add sucrose solution dissolved in water for injection, 55'C water bath, magnetically stir for 1 hour, ultrasonically disperse, homogenize with high pressure homogenizer, extrude with 0.2 micron polycarbonate membrane, sterile filter 0.22 micron PVDF membrane, dispense Into the vial, freeze-dried, argon in the vial, sealed with a gland.

The average particle diameter of the sample before lyophilization was 178.5 nm and the encapsulation efficiency was 92.4% as measured in the same manner as in Example 1. The pharmacologically active ingredients and main raw materials used in the above examples are as follows: Various phospholipids were purchased from LIPOID GmbH, cholesterol was purchased from Henan Zhengzhou Liwei Bio-Industry Co., Ltd., sodium cholesteryl sulfate and potassium sulphate sulfate were purchased from Xi'an Libang Pharmaceutical Co., Ltd. Ltd., paclitaxel was purchased from Shanghai Dyson Biomedical Co., Ltd. Sucrose was purchased from Merck China.

Example 1 Stability test of paclitaxel liposome composition

This test was conducted to examine the storage stability and the use stability of the liposome samples prepared in the examples.

The dried paclitaxel liposome compositions prepared in Examples 1, 2, and 3 were stably stored at 2 to 10 ° C for a long period of time, and the appearance thereof was observed. After reconstitution with water for injection, the results were observed under a microscope, and the results are shown in Table 1; The solution was stored at room temperature for 12 hours, and observed at regular intervals. The results are shown in Table 2. From the results, it was found that the paclitaxel lipid film was stored at 2 to 10 ° C for 12 months, and did not aggregate after reconstitution, and no paclitaxel crystals were precipitated; after reconstitution, it was left for 12 hours, no aggregation occurred, and no paclitaxel crystals were precipitated. Table 1 Paclitaxel liposome low temperature (2~10 °C) placement stability test results

Sample when placed

Appearance Microscopy after reconstitution

Source (month)

0 type white loose mass No crystal precipitation after dissolution, uniform appearance, no aggregation

Class 1 white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation

3. Class 3 white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation.

1

6 kinds of white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation

12 kinds of white loose masses, no crystals are precipitated after dissolution, the appearance is hooked, no aggregation

Class 3 white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation

2

12 kinds of white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation

Class 3 white loose masses, no crystals precipitated after dissolution, uniform appearance, no aggregation 3

12 kinds of white loose masses have no crystal precipitation after dissolution, uniform appearance, no aggregation 4 Table 2 Results of stability test at room temperature after reconstitution of paclitaxel liposomes

Example 8 Acute Toxicity Test of Paclitaxel Liposomes

This example is intended to investigate the immediate toxicity and multi-drug tolerance of the paclitaxel liposomes of the present invention after administration in a conventional manner.

The paclitaxel liposomes prepared in Example 1 were reconstituted with water for injection. Test animals: Kunming female mice, weighing 18 to 20 g, were randomly divided into 4 groups, 5 in each group, and the negative group was given the corresponding volume in the highest volume of the test group. Each group was administered intravenously on days 1, 4, and 7.

Test results: (1) Immediate toxic side effects observation: Immediately after administration of each group of mice, no obvious side effects were observed. (2) Change in body weight of mice: The body weight of the mice did not change much after administration of the paclitaxel group, and the body weight of the mice decreased after the third administration, and the body weight gradually increased thereafter. The mice in the blank solvent group gradually increased in body weight.

The specific results are shown in Table 3. Table 3 Changes in animal body weight in paclitaxel liposome acute toxicity test

Example 9 Antitumor effect test

A preliminary anti-tumor test was carried out using the paclitaxel liposome composition prepared in Example 1. The experimental animals were C ₅₇ BL/6 black mice, male, weighing 19-22 g, randomized into groups of 10 animals each. The B16 melanoma source was taken, and the tumor cell suspension was prepared by the homogenization method with physiological saline 1:6, and the B16 melanoma was inoculated subcutaneously. The tumor was injected into the tail vein 24 hours after the tumor inoculation, and the immediate reaction was observed. Weight changes. At the end of the experiment, the tumor was weighed and the tumor inhibition rate was calculated as follows.

Tumor inhibition rate%=[(Control mean tumor weight-average tumor weight of the administration group) / Control group average tumor weight] * 100% Test results: (1) Immediate reaction observation: No effect of paclitaxel liposome after administration Immediately obvious toxic and side effects. After intravenous administration of the mice in each group (the injection rate was 1 ml/min), the mice were in a semi-coma state and gradually recovered in about 15 minutes. (2) There was no significant change in body weight of each group of animals. (3) There was no significant difference in the antitumor effect between paclitaxel liposomes and Taxol at the same total dose (see Table 4). Table 4 Paclitaxel liposome different dosing regimen for mouse melanoma B16 (subcutaneous inoculation) efficacy test group animals (only) tumor inhibition rate dosing regimen

Sample IJ amount (mg/kg) start/end (%)

50 Day 1 and 7 intravenous administration 10/10 64.87 Paclitaxel

33.3 Intravenous administration on days 1, 5 and 9 10/10 65.38 liposome

25 Day 1, 4, 7 and 10 days of intravenous administration 10/10 65.77

33.3 Day 1, 5, 9 intravenous injection 10/10 68.11 Taxol

25 Day 1, 4, 7 and 10 days of intravenous administration 10/10 69.18

Claims

Rights request

A stable liposome characterized in that the liposome-forming lipid comprises a hydrogenated saturated phospholipid, cholesterol and a 3-substituted derivative of cholesterol, and the active ingredient is encapsulated in the liposome.

The liposome according to claim 1, wherein the 3-substituted derivative of the cholesterol is selected from the group consisting of sodium cholesteryl sulfate or potassium cholesterate. '

The liposome according to claim 2, wherein the active ingredient is a taxane.

The liposome according to claim 3, wherein the taxane-like substance is paclitaxel.

The liposome according to claim 4, wherein the molar ratio of the paclitaxel to the hydrogenated saturated phospholipid is 2 to 8.5:100.

6. The liposome according to any of the preceding claims, wherein the hydrogenated saturated phospholipid is selected from the group consisting of hydrogenated saturated soybean phospholipids, hydrogenated saturated lecithin, dipalmitoylphosphatidylcholine and distearoyl phospholipids. One or more of choline.

The liposome according to any one of the preceding claims, wherein the molar ratio of the hydrogenated saturated phospholipid, cholesterol and cholesterol sulfate sodium or potassium salt is: 100:17~50:0.04~5 .

The liposome according to claim 7, wherein the hydrogenated saturated phospholipid, the molar ratio of cholesterol to cholesterol sulfate sodium salt or potassium salt is: 100: 35 to 45: 0.5 to 2.

The liposome according to any of the preceding claims, wherein the liposome further comprises natural or synthetic vitamin E as an antioxidant, and the weight ratio to the hydrogenated saturated phospholipid is from 0.13 to 1.25: 100.

A liposome according to any of the preceding claims wherein the liposomes have an average particle size in the range of from 100 nanometers to 300 nanometers.

A composition comprising the liposome of any one of claims 3 to 10, characterized in that the composition further comprises a lyoprotectant in the form of a lyophilizate. ;

The liposome composition according to claim 11, wherein the lyoprotectant is selected from the group consisting of sucrose, lactose, maltose, and glucose.

The liposome composition according to claim 12, wherein the lyoprotectant is 1.5 to 10 times the weight of the hydrogenated saturated phospholipid.

The method for preparing a liposome composition according to any one of claims 11 to 13, wherein the active ingredient and the film-forming lipid, if necessary, and an antioxidant are stirred and dissolved in ethanol in proportion. Constant temperature 50 ° C ~ 65O, remove the solvent under reduced pressure, form a film, add an aqueous solution of lyophilized excipients, hydrated, sonicated or slurried or extruded to a fineness of 0.1~

Between 5.0 micrometers, the bacteria were sterilized by filtration through a 0.22 micron microporous membrane, subpacked, and frozen to obtain a white block. Paclitaxel liposome composition.

The use of the liposome composition according to any one of claims 11 to 13 for the preparation of an anticancer drug, wherein the cancer species are ovarian cancer, ovarian metastatic cancer, breast cancer and non-small cell lung cancer.

The use according to claim 15, wherein the liposome composition is reconstituted with water for injection before use, and then diluted with a 5% dextrose solution, which is used by intravenous or intraperitoneal injection or tumor. Body injection.

17. Use according to claim 16, characterized in that the liposome composition can be used in combination with cisplatin for the treatment of cancer.

18. A method of treating cancer in a human comprising administering to a cancer patient a liposome composition according to any one of claims 11 to 13 comprising an effective amount of paclitaxel, the type of cancer comprising ovarian cancer, ovarian metastatic cancer , breast cancer and non-small cell lung cancer.

19. The method of treating cancer in a human of claim 18, wherein the method of administering to a cancer patient comprises intravenous injection or intraperitoneal injection or intratumoral injection.

20. A method of treating cancer in a human of claim 18 which comprises administering said liposome composition in combination with an effective amount of cisplatin.