CN107669637B - Artemether liposome for injection and preparation method and application thereof - Google Patents
Artemether liposome for injection and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Abstract
The invention provides an artemether liposome for injection, which is prepared by adopting an ethanol injection method, has simple preparation process and can be applied to industrial production, and the artemether liposome prepared by selecting proper material components and adopting a proper preparation process has small particle size (150-200 nm), uniform particle size distribution and encapsulation rate of more than 90 percent. The artemether liposome is slowly released in vivo, so that adverse reaction caused by overhigh concentration of the medicament in the initial injection stage is avoided. Meanwhile, the inventor finds that the artemether liposome prepared by the method has high stability, high bioavailability, good redissolution property and long storage time, and meets the requirement of medication.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, and in particular relates to an artemether liposome for injection and a preparation method and application thereof.
Background
Artemisinin and its derivatives are sesquiterpene lipid compounds extracted from leaves of artemisia annua, the drugs are high-efficiency and quick-acting plasmodium erythrocytic internal phase killing agents, and are mainly used for symptom control of vivax malaria and plasmodium falciparum and treatment of chloroquine-resistant strains at present; recent researches find that artemether has stronger antitumor activity and has the effect of inhibiting and killing various tumor cells, so that the antitumor activity of artemether is a research hotspot in newly found pharmacological action. Artemether is 10-beta-methyl dihydro derivative of artemisinin, and its molecular formula is C16H26O5. The shape is white crystal or crystalline powder. The antimalarial activity of the artemether is 10-20 times stronger than that of the artemisinin, and the recent recurrence rate of the artemether is lower than that of the artemisinin. The existing artemether dosage forms on the market mainly comprise capsules, tablets and oil-soluble injections. Due to the poor water solubility of artemether, the artemether has slow absorption in the digestive tract, low bioavailability, short biological half-life and difficult maintenance of effective blood concentration. The oil-soluble injection is easy to cause adverse reactions such as allergy and the like, injection pain and stimulation to injection parts. Therefore, research and development of a safe and efficient novel artemether drug delivery system can improve the bioavailability, and artemether can be clinically appliedThe application in (1) has positive significance.
The liposome is a closed vesicle formed by encapsulating a medicament in a lipid bilayer consisting of phospholipid and cholesterol, has the tropism of a lymphatic system, and belongs to a novel dosage form of a targeted drug delivery system. The liposome has good biocompatibility and is easy to be fused into cells to further release the medicine, so that the liposome can keep higher blood concentration in specific tissues, can change the tissue distribution of the medicine in vivo, ensures that the medicine is mainly gathered in tissues and organs such as liver, spleen, lung, bone marrow and the like, and can further improve the curative effect of the medicine and reduce the toxic and side effect of the medicine. After the medicine is prepared into liposome, the active groups of the medicine can be protected from being degraded by enzymes in vivo, so that the circulation time of the medicine in blood can be prolonged.
However, despite the advantages of the liposome, long-term practical research shows that the liposome has poor stability, the liposome prepared by the general method is easy to aggregate and fuse, and the effective period is short; due to the fact that liposome properties are not stable enough, the content of the liposome is easy to leak, in order to improve the storage stability of the liposome, freeze drying is usually carried out at present, water is added for redissolving before use to form liposome suspension, but liposome vesicles are easy to damage after the liposome is freeze-dried into freeze-dried powder, the drug leakage phenomenon occurs, the liposome is difficult to recover to the state before freeze drying after freeze drying, and the content leaks more or less, so that a freeze-drying protective agent needs to be added, but the addition of the substance can affect the use safety of the liposome, meanwhile, different drugs and the bioavailability of the liposome prepared by different preparation methods are different, aiming at different drugs, the entrapment rate and the drug loading rate of the liposome prepared by some drugs are greatly different, so that the drug loading of the liposome prepared by some drugs is extremely low, and the effect of the drugs is difficult to exert.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor provides the artemether liposome for injection, which not only can effectively improve the bioavailability of the artemether liposome, but also can enhance the thermal stability of artemether and prolong the effective period of artemether.
Specifically, the invention relates to the following technical scheme:
the invention discloses a first aspect of artemether liposome for injection, which is prepared by selecting the following components in specific weight ratio: artemether, soybean phospholipid and cholesterol can form artemether liposome for injection with excellent quality.
Specifically, the invention provides an artemether liposome for injection, which is prepared from the following components in parts by weight:
1 part of artemether, 6-8 parts of soybean phospholipid and 1-2 parts of cholesterol;
further preferably, the artemether liposome for injection is prepared from the following components in parts by weight:
1 part of artemether, 6 parts of soybean phospholipid and 1.5 parts of cholesterol;
as the phospholipid for forming the liposome, natural phospholipid and synthetic phospholipid can be selected, and in the present invention, natural phospholipid and synthetic phospholipid can be used as the phospholipid for forming the liposome as the pharmaceutically active ingredient. In the present invention, artemether, which is a pharmaceutically active ingredient, has good lipid solubility and poor water solubility. Aiming at the characteristics of artemether, the inventor finds that soybean phospholipid is particularly suitable to be used as a basic phospholipid film-forming material through research.
Soybean lecithin is a natural phospholipid, and has high content, easy availability and low cost. The soybean lecithin has high phase transition temperature and is easy to form a stable liposome membrane. When other phospholipid components such as egg yolk lecithin, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, etc. are used, it is difficult to form a liposome having excellent quality, and the liposome has poor properties such as encapsulation efficiency, stability, and leakage rate.
In particular, in the artemether liposome for injection of the present invention, the soybean phospholipids are used in an amount of 6 to 8 parts by weight relative to 1 part by weight of artemether. If the amount of soybean phospholipids is less than 6 parts by weight, stable liposomes cannot be formed; on the other hand, if the amount of soybean phospholipids is more than 8 parts by weight, the encapsulation efficiency of artemether as a pharmaceutically active ingredient is decreased, and the quality and therapeutic effect of the injection are decreased.
In the present invention, cholesterol is used to modulate the membrane stability of liposomes. Cholesterol is an amphiphilic molecule that binds to soy phospholipids, preventing them from agglomerating into a crystalline structure. Cholesterol is incorporated into the soybean phospholipid bilayer and acts like a "buffer" to regulate the "fluidity" of the membrane structure. When the temperature is lower than the phase transition temperature, the cholesterol can reduce the orderly arrangement of the membrane and increase the fluidity; above the phase transition temperature, cholesterol can increase the ordered arrangement of the membrane, thereby reducing the fluidity of the membrane. Cholesterol can solidify the liposome bilayer membrane, thereby reducing the generation of free radicals, reducing the oxidation level and obviously enhancing the stability of the liposome. Research shows that the stability of the liposome has close correspondence with the bioavailability. The higher the stability, the higher the bioavailability.
On the other hand, the inventor researches and discovers that in the artemether liposome for injection, the encapsulation efficiency of the artemether liposome is higher when the soybean phospholipid is used in an amount of 6-8 parts by weight and the cholesterol is used in an amount of 1-2 parts by weight relative to 1 part by weight of artemether. Particularly, when the mass ratio of artemether, soybean phospholipid and cholesterol is 1:6:1.5, the obtained liposome has the best stability and the highest entrapment rate.
It has been found that when using the above-mentioned specific amounts of artemether, soya lecithin and cholesterol, artemether liposomes of good quality can be obtained, which have high encapsulation efficiency and stability, low toxicity and high bioavailability.
In another aspect of the invention, a preparation method of artemether liposome is disclosed, which comprises the following steps:
dissolving artemether, soybean phospholipid and cholesterol in absolute ethyl alcohol according to a certain proportion, performing ultrasonic dissolution to prepare a lipid solution, then injecting the lipid solution into Phosphate Buffer Solution (PBS) at the temperature of 40-45 ℃, stirring to remove the ethanol after the injection is finished, and then performing ultrasonic treatment on a filter membrane to obtain the artemether liposome liquid preparation.
Wherein the mass volume ratio of the artemether, the soybean phospholipid, the cholesterol, the absolute ethyl alcohol and the phosphate buffer solution is 1:6-8:1-2:0.3-0.4:0.8-1.2 (g: g: L: L);
further preferably, the mass volume ratio of the artemether, the soybean phospholipid, the cholesterol, the absolute ethyl alcohol and the phosphate buffer solution is 1:6:1.5:0.3:1.2 (g: g: g: L: L);
the ultrasonic frequency is 30-50KHz (preferably 40KHz) when ultrasonic dissolution is carried out, and the stirring rotating speed is 700 and 800rpm (preferably 800rpm) when stirring is carried out to remove ethanol; the ultrasonic frequency is 30-50KHz (preferably 40KHz) when ultrasonic treatment is carried out;
preferably, the pore size of the filter membrane is 0.22 μm;
preferably, the phosphate buffer has a pH of 6.5;
in another aspect of the invention, the application of the artemether liposome for injection prepared by the preparation method in preparing a medicament for targeted treatment of liver cancer tumor cells is disclosed.
As mentioned above, artemether is traditionally used for symptom control of vivax malaria and malignant malaria and treatment of chloroquine-resistant strains, however, recent research shows that artemether has strong antitumor activity and has inhibition and killing effects on various tumor cells, and therefore, the antitumor activity of artemether is a research hotspot in newly discovered pharmacological action. The liposome has slow release effect and targeting effect, so the artemether liposome prepared by the liposome can effectively improve the tumor treatment effect. However, the artemether liposome prepared by the invention has poor targeting effect and accumulation effect on brain cancer tumors and lung cancer tumors, which also indicates the extreme complexity of the pharmaceutical preparation in the process of treating tumor and cancer.
The invention adopts an ethanol injection method to prepare artemether liposome, the preparation process is simple, the method can be applied to industrialized production, the invention selects safe and nontoxic absolute ethanol as a solvent to dissolve lipid materials, the influence of the dosage on the stability after freeze drying is surprisingly found to be great, the inventor finds that, in a proper dosage range, the absolute ethanol can induce the acyl chain of the flowing phospholipid to disorderly form an interlaced gel phase, when in the freeze drying process, a large amount of water molecules in the lipid bilayer sublime to destroy the gel phase structure, so that the lipid bilayer fuses and the drug leaks, and part of water molecules in the liposome vesicle are replaced by the ethanol molecules by controlling the content of the absolute ethanol, and the artemether is easier to dissolve in the ethanol, so that artemether particles are wrapped in the ethanol, and are separated out to form a network-like framework structure in the liposome during the freeze drying process, the drug leakage is reduced, and meanwhile, the damage of water molecules to a gel phase structure is reduced, so that the stability of the artemether liposome is maintained, the re-solubility of the artemether liposome is remarkably improved, the use of a freeze-drying protective agent is reduced, and the negative influence of the freeze-drying protective agent on the artemether liposome is reduced; meanwhile, the inventor finds that ultrasonic treatment has great influence on the particle size control and the entrapment rate of the liposome prepared by the method, and the pH of the buffer solution is controlled by adopting a phosphate buffer solution, so that the liposome solution is diluted, and the final product is in a more stable environment to play a buffering role; meanwhile, the inventor surprisingly finds that the targeting effect and the accumulation effect of the artemether liposome on the liver cancer tumor are obviously improved after the artemether liposome is treated by the phosphate buffer solution immersion liquid with the pH value of 6.5;
according to the invention, by selecting appropriate material components and adopting an appropriate preparation process, the prepared artemether liposome has small particle size (150-200 nm), uniform particle size distribution and encapsulation rate of more than 90%; as artemether is coated on the phospholipid membrane of the liposome, the encapsulation plays a role in enhancing the stability, avoids the direct contact between the drug and the vessel wall and reduces the vascular irritation of the drug. In addition, the artemether liposome is slowly released in vivo, so that adverse reactions caused by overhigh concentration of the medicament in the initial injection stage are avoided. Meanwhile, the inventor finds that the artemether liposome prepared by the method has high stability, high bioavailability, good redissolution property and long storage time, and meets the requirement of medication.
Drawings
FIG. 1 is a picture of fresh artemether liposomes prepared in example 1 of the present invention.
FIG. 2 is a graph showing the particle size and particle size distribution of artemether liposomes prepared in example 1 of the present invention.
FIG. 3 is a graph showing the potential distribution of artemether liposomes prepared in example 1 of the present invention.
FIG. 4 is a transmission electron micrograph of artemether liposomes prepared in example 1 of the present invention.
FIG. 5 is a graph showing the variation of the particle size of artemether liposomes prepared in example 1 of the present invention in different media.
FIG. 6 is a comparison of the in vitro release profiles of artemether solubilization solution and artemether liposomes prepared in example 1 of the present invention.
FIG. 7 shows the results of the hemolysis test of artemether liposomes prepared in example 1 of the present invention (negative control No. 1, artemether liposomes of different concentrations from No. 2 to No. 6, and positive control No. 7);
FIG. 8 is a result of the hemolysis rate of artemether liposomes prepared in example 1 of the present invention;
FIG. 9 is a photograph of pathological sections of major organs of each group of mice after administration of physiological saline, a blank liposome group, an artemether solubilization solution, and artemether liposomes prepared in example 1 of the present invention.
FIG. 10 is a graph of the drug concentration in the plasma of each group of mice as a function of time after administration of the artemether solubilized solution and the artemether liposomes prepared in example 1 of the present invention.
FIG. 11 is a graph of tumor volume versus time for groups of mice administered with saline, blank liposome group, artemether solubilization solution, and artemether liposomes prepared in example 1 of the present invention.
FIG. 12 is a photograph showing the appearance of tumors in each group of mice after administration of physiological saline, a blank liposome group, an artemether solubilization solution, and artemether liposomes prepared in example 1 of the present invention.
FIG. 13 is a photograph of pathological sections of tumors in each group of mice after administration of physiological saline, a blank liposome group, an artemether solubilization solution, and artemether liposomes prepared in example 1 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, artemether has poor water solubility, resulting in slow absorption in the digestive tract, low bioavailability, short biological half-life and difficult maintenance of effective blood concentration. The oil-soluble injection is easy to cause adverse reactions such as allergy and the like, injection pain is caused, the injection part is stimulated, and the compliance of a patient is poor. Therefore, the research and development of a safe and efficient novel artemether drug delivery system can improve the bioavailability, and has positive significance for the clinical application of artemether.
In view of the above, in one embodiment of the present invention, an artemether liposome for injection is provided, which is prepared from the following components in parts by weight:
1 part of artemether, 6-8 parts of soybean phospholipid and 1-2 parts of cholesterol;
further preferably, the artemether liposome for injection is prepared from the following components in parts by weight:
1 part of artemether, 6 parts of soybean phospholipid and 1.5 parts of cholesterol;
in another embodiment of the present invention, a method for preparing artemether liposome is disclosed, which comprises the following steps:
dissolving artemether, soybean phospholipid and cholesterol in absolute ethyl alcohol according to a certain proportion, performing ultrasonic dissolution to prepare a lipid solution, then injecting the lipid solution into Phosphate Buffer Solution (PBS) at the temperature of 40-45 ℃, stirring to remove the ethanol after the injection is finished, and then performing ultrasonic treatment on a filter membrane to obtain the artemether liposome liquid preparation.
In another embodiment of the present invention, the weight volume ratio of artemether, soybean phospholipid, cholesterol, absolute ethyl alcohol and phosphate buffer solution is 1:6-8:1-2:0.3-0.4:0.8-1.2 (g: g: g: L: L);
in another embodiment of the invention, the mass-to-volume ratio of artemether, soybean phospholipid, cholesterol, absolute ethyl alcohol and phosphate buffer is 1:6:1.5:0.3:1.2 (g: g: g: L: L);
in another embodiment of the present invention, the ultrasonic frequency is 30-50KHz (preferably 40KHz) when ultrasonic dissolution is performed, and the stirring speed is 700-800rpm (preferably 800rpm) when ethanol is removed by stirring; the ultrasonic frequency is 30-50KHz (preferably 40KHz) when ultrasonic treatment is carried out;
preferably, the pore size of the filter membrane is 0.22 μm;
preferably, the phosphate buffer has a pH of 6.5;
in another specific embodiment of the invention, the application of the artemether liposome for injection prepared by the preparation method in preparing a medicament for targeted therapy of liver cancer tumor cells is disclosed, and the medicament for targeted therapy of liver cancer tumor cells is an injection.
The invention is further illustrated by the following examples.
Example 1
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 800rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 2
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 4ml of absolute ethyl alcohol 30KHz, uniformly mixing, slowly injecting the mixed solution into 10ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 700rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 3
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by 4ml of absolute ethanol at 50KHz, uniformly mixing, slowly injecting the mixed solution into 10ml of PBS heated to 45 ℃, stirring by an electronic constant speed stirrer at 700rpm until the absolute ethanol is completely removed, ultrasonically treating the obtained suspension (30KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 4
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethanol 40KHz, uniformly mixing, slowly injecting the mixed solution into 10ml of PBS heated to 45 ℃, stirring by using an electronic constant speed stirrer at the speed of 700rpm until the absolute ethanol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 5
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 40 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 700rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (30KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 6
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 8ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 700rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Example 7
The preparation process comprises the following steps:
dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz in an ultrasonic mode, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 800rpm until the absolute ethyl alcohol is completely removed, and performing ultrasonic treatment (30KHz) on the obtained suspension and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Experimental example 8
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 800rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Experimental example 9
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by 7ml of absolute ethanol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 45 ℃, stirring by an electronic constant speed stirrer at the speed of 800rpm until the absolute ethanol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
Experimental example 10
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount in 3ml of absolute ethanol 40KHz, mixing, slowly injecting the mixed solution into 12ml of citrate buffer solution heated to 45 ℃ by using a microsyringe, stirring at 800rpm by using an electronic constant speed stirrer until the absolute ethanol is removed, and ultrasonically treating the obtained suspension (40KHz)
Then, the artemether liposome is obtained by passing through a 0.22 mu m microporous filter membrane.
Experimental example 11
The preparation process comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescribed amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of monopotassium phosphate-sodium hydroxide buffer solution heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 800rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension (40KHz), and passing through a 0.22 mu m microporous filter membrane to obtain the artemether liposome.
The encapsulation efficiency (encapsulation efficiency I), drug loading rate and particle size of artemether liposome in the liposome suspension, and the liposome (encapsulation efficiency II) after freeze-drying and hydration and reconstitution with injection water were measured for the liposomes of examples 1 to 7 and experimental examples 8 to 11 as shown in the following table.
Stability quality investigation
The liposomes prepared in the above examples 1 to 7 and examples 8 to 11 were examined and counted for their long-term stability with reference to the appendix of the Chinese pharmacopoeia, and the results were shown in the following table:
as can be seen from the above table, when the optimal ratio of the raw materials of the invention is separated or the raw materials of the invention are replaced (examples 8 and 9), the encapsulation efficiency and drug-loading capacity are obviously reduced, and the stability experiment is not ideal, especially in example 9, only the amount of absolute ethyl alcohol is increased, the effect of reducing the encapsulation efficiency of artemether liposome in liposome suspension is not obvious, but the encapsulation efficiency of artemether liposome after being redissolved after freeze-drying is obviously reduced; meanwhile, the inventor selects and uses a citrate buffer solution and a potassium dihydrogen phosphate-sodium hydroxide buffer solution in addition to a PBS buffer solution in the aspect of buffer solution selection, and the two buffer solutions are equivalent to or even superior to the technical scheme of the invention adopting the PBS buffer solution in the aspects of entrapment rate, drug loading capacity and stability experiments, but the inventor finds that the targeting effect and the accumulation effect of the artemether liposome prepared by the experiment examples 10 and 11 on liver cancer tumor are both obviously lower than those of the artemether liposome (p is less than 0.05) in the example 1 in the experiment of treating mouse liver cancer tumor, which shows that the targeting effect and the accumulation effect of the prepared artemether liposome are greatly influenced by the phosphate buffer solution with the pH of 6.5 on liver cancer tumor cells, because the liposome of the invention is passively targeted, the target effect is realized by multiple factors, the artemether liposome comprises an EPR effect, a tumor region microenvironment, a reduction environment in tumor cells and the like, and the factors are closely related to the particle size, charge distribution, liposome composition, pH and the like of the liposome, and the inventor considers that the conditions of the artemether liposome of the invention treated by phosphate buffer solution with pH of 6.5 better meet the target positioning of liver cancer tumors, and simultaneously, the liposome is more favorable for accumulation in the liver cancer tumor cells, thereby realizing the high-efficiency treatment of the artemether on the liver cancer tumor cells.
Experimental part
1. Investigation of stability of artemether liposome in different media
The particle size change of the artemether liposome is taken as an evaluation index, the stability of the artemether liposome in 10% plasma-normal saline solution and pH7.4PBS solution is respectively examined, and the test result is shown in figure 5, and after the artemether liposome suspension and 10% plasma are incubated, the particle size of the artemether liposome suspension does not change obviously in the first 24 hours. The artemether liposome has certain stability in the two mediums and can maintain a relatively complete structure in vivo. After 24 hours, the artemether liposome has no obvious change in the PBS particle size and has good stability. In 10% of blood plasma, the particle size is obviously increased, the maximum particle size is close to 1100nm, and floccules can be seen with naked eyes. The results show that under the condition, the lipid bilayer structure of the artemether liposome is damaged, and the drug can be fully released.
2. In vitro release assay
The in vitro release of liposomes of the invention was studied with reference to dynamic membrane dialysis. Taking 3 parts of artemether solubilization solution and liposome of embodiment 1 of the invention, each part containing about 2mg of artemether, placing the artemether into a pretreated dialysis bag, fastening the bag opening, placing the bag opening into a dialysis tube containing 25mL of release medium, placing the sample into a constant temperature oscillator, controlling the temperature to be (37 +/-1) DEG C and the rotating speed to be 100rpm, periodically sucking 1.0mL of dialysate according to a set time point, timely supplementing equivalent release medium, measuring the concentration of the drug, and calculating the cumulative release rate Q (%). In vitro release curves are plotted with Q mean as ordinate and release time (t) as abscissa in fig. 6.
As can be seen from FIG. 6, in PBS (pH7.4), the cumulative release percentage of artemether solubilization solution group in the first 8h reaches more than 80%, while the release of artemether liposome group has no obvious burst, at 8h, artemether liposome group releases (50.46 + -2.03)%, at 48h, the cumulative release percentage reaches (79.35 + -2.10)%, and the release speed is obviously slower than that of artemether solubilization solution group.
3. Safety test
3.1. Hemolysis test
7 test tubes are respectively numbered from 1 to 7, wherein the tube 1 is a negative control tube and has no hemolysis phenomenon; no. 7 is a positive control tube, and hemolysis is caused; the No. 2-6 tube is a sample tube. 2% erythrocyte suspension, liposome suspension, normal saline and distilled water were added in sequence as shown in Table 1, mixed uniformly, and incubated at 37 deg.C for 3 h. After the incubation was completed, the sample solution was placed in a centrifuge and centrifuged at 3000rpm for 10 min. The supernatant was observed to determine whether hemolysis occurred. Detecting the hemolysis rate under ultraviolet to judge the hemolysis degree. Hemolysis was observed in 7 tubes and the results are shown in FIG. 7.
TABLE 1 hemolytic assay design
The hemolysis of each group was judged by visual observation and uv spectrophotometry.
The hemolysis of 7 tubes was visually observed, and the results are shown in FIG. 7; from the results of fig. 7, it can be seen that the suspension of artemether liposomes had no significant hemolysis under visual observation.
The hemolysis rate of artemether liposome (ARM-Lips) measured by ultraviolet spectrophotometry is shown in FIG. 8; from the above results, the hemolysis percentage of artemether liposome at each concentration was 5% less, indicating that it can be safely used for intravenous injection.
3.2. Pathological section
In mice for observing pathological changes, the mice are divided into four groups, and each group comprises 3 mice, namely an NS group, a blank-Lips group, an artemether solubilization solution (ARM-Sol) group and an ARM-Lips group. The administration dose was 20mg/kg, once every 2 days, for 10 consecutive days. After 10 days, after the mice are sacrificed, HE staining is carried out on normal organ tissues of the mice so as to observe the toxic and side effects of each group of preparations on the normal organ tissues; as can be seen from FIG. 9, after the administration is finished, the morphology of each main organ of the mice in the artemether liposome group has no obvious pathological changes compared with the physiological saline group. Therefore, the liposome is used as a carrier of artemether, has low toxicity and good biocompatibility.
4. Pharmacokinetic testing
66 Kunming mice are randomly divided into 2 groups, namely an artemether solution group and an artemether liposome group. The administration dose is 20mg/kg, and the administration mode is tail vein injection. At 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10h after administration, 500. mu.L of blood was collected from the eyeball of the mouse, and after blood collection, the mouse was killed by removing the neck. Placing the plasma in EP tube rinsed with heparin, centrifuging (12000 rpm) for 15min, taking out supernatant, storing in refrigerator at-20 deg.C, and testing; the DAS2.0(Drug And Statistics for Windows) pharmacokinetic program processed the measurement results of Drug concentration in the plasma of mice to calculate pharmacokinetic parameters, wherein the mean plasma concentration versus time curve is shown in fig. 10.
The measurement results of the blood concentration were processed with DAS2.0(Drug And Statistics for Windows) pharmacokinetic software to calculate pharmacokinetic parameters, And the results are shown in table 2.
Table 2 kinetic parameters of two formulations of artemether in mouse plasma
As can be seen from the above table and FIG. 10, ARM-Lips significantly increased circulation time in vivo, with 8.375 fold increase in t1/2 β, 3.38 fold increase in MRT, and 3.11 fold increase in AUC, respectively, compared to ARM-Sol.
5. Pharmacodynamic test
Mice were inoculated with H22 hepatoma tumor cells in the left axilla. H22 cells were selected at logarithmic growth phase, cell viability was determined to be > 95%, washed with PBS, suspended and counted. Mice were fixed, their left underarm disinfected with alcohol, and inoculated with 0.1mL of H22 cell suspension (2X 10)7/mL). Measuring the length and the short diameter of the tumor by a vernier caliper, and applying the formula of tumor volume (L multiplied by W)2) The tumor volume was calculated.
When the tumor volume is more than 100mm3In the meantime, 41 selected from 60 tumor model mice are preferably selected to have similar tumor volume and small weight differenceIn 1g of mice.
In mice observed for volume change, the total volume was divided into 4 groups of 8 mice, each group was a normal saline group (NS), a blank liposome group (blank-Lips), an artemether solubilization solution group (ARM-Sol), and an artemether liposome group (ARM-Lips). The administration dose was 20mg/kg, once every 2 days for 10 consecutive days, and the body weight of the mice and the major and minor diameters of the tumors were measured one day after the administration to calculate the tumor volume of the mice. The tumor volume and the body weight of the mice were plotted. After 10 days, the mice were sacrificed, the tumor bodies were detached, and their weights were weighed.
In mice observing pathological changes, the mice are divided into four groups, each group comprises 3 mice, and the groups are respectively an NS group, a blank-Lips group, an ARM-Sol group and an ARM-Lips group. The mode of administration, dosage and time are the same as described above. After 10 days, after the mice were sacrificed, HE staining was performed on the tumor and normal organ tissues to observe the structural difference of the tumor and the toxic and side effects on the normal organ tissues in each group of the preparations.
Mice were sacrificed on day 11 post-dose, and the dissected tumor bodies were weighed and photographed, and the results are shown in fig. 12.
On the 11 th day after administration, each group of tumor bodies are stripped and observed by naked eyes, the tumor volumes of the blank liposome group and the normal saline group are larger, the artemether solution group is second, the artemether liposome group is the smallest, and the tumor weights are sequentially arranged from large to small: blank liposome group > physiological saline solution group > artemether liposome group. The results show that the liposome group and the solution group can inhibit the growth of tumors to different degrees, and the tumor weight of the liposome group is smaller than that of the solution group, which indicates that the anti-tumor effect of the artemether liposome is better than that of the artemether solution group.
The difference of the tumor structure after the artemether liposome is administrated is examined by pathological sections, and the results are respectively shown in figure 13. As can be seen from fig. 13, after the administration was completed, the tumor cells in the normal saline group and the blank liposome group had intact structures, the nuclear division image was obvious, and most of the tumor cells were in a state of vigorous proliferation and division; in the artemether solution group, a small part of tumor cell structures are damaged, and most of tumor cells still show an actively proliferating state; in the artemether liposome group, the tumor cell structure is seriously damaged, a large area of necrosis is shown, inflammatory cells infiltrate, and the number of nuclear divisions is reduced.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (1)
1. An application of artemether liposome for injection in preparing medicine for target treatment of hepatocarcinoma and tumor cell is provided;
the preparation method of the artemether liposome for injection comprises the following steps:
ultrasonically dissolving artemether, soybean phospholipid and cholesterol in a prescription amount by using 3ml of absolute ethyl alcohol 40KHz, uniformly mixing, slowly injecting the mixed solution into 12ml of PBS heated to 45 ℃ by using a microsyringe, stirring by using an electronic constant speed stirrer at the speed of 800rpm until the absolute ethyl alcohol is completely removed, ultrasonically treating the obtained suspension by using 40KHz, and passing through a 0.22 mu m microporous filter membrane to obtain artemether liposome;
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| CN106474064A (en) * | 2015-08-27 | 2017-03-08 | 昆药集团股份有限公司 | A kind of Artemether nanometer liposome and preparation method and application |
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