CN113908123A - A kind of micelle containing taxane drugs, its preparation method and application - Google Patents
A kind of micelle containing taxane drugs, its preparation method and applicationDownload PDFInfo
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Abstract
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a micelle containing taxane medicines, and a preparation method and application thereof.
Background
Taxanes are a large group of natural drugs obtained from plants, and belong to diterpenes in structural classification. Both cabazitaxel and docetaxel belong to taxane drugs, are fat-soluble compounds, and have limited clinical application due to the defects of poor water solubility, short half-life, high toxicity and the like. At present, both docetaxel injection and cabazitaxel injection on the market at home and abroad are prepared by dissolving drugs in tween-80, and the Clinical application requires strict use of a special injection solvent for dilution, and strict operation requirements, and a special infusion device is also required during infusion, so that the use method is complicated, and the preparation contains a large amount of tween-80, so adverse reactions such as hemolysis, Allergy and the like are very easy to cause, and dexamethasone and other drugs are required to be taken in advance for prevention, so that the Clinical medication is inconvenient, and the medication safety is low (analls of Allergy and Clinical immunization, 2005,95(6): 593-. This problem has not been solved well to date.
Liposomes containing taxanes are known in the prior art, but they also have a number of disadvantages. Such as: the preparation process of the liposome is complex, the composition of a plurality of lipid components (at least two lipid components) is needed, and special equipment and devices are needed for controlling the particle size; easy flocculation in the storage process, etc.
The entrapment rate of the cabazitaxel liposome prepared by Zhangli and the like of Jiangsu university is only about 86%, the particle size change before and after freeze-drying is large, the particle size is increased from 68nm to 108nm, and the instability of the liposome after re-dissolution is increased. (preparation of Zhangli. Cabazitaxel lyophilized liposome and pharmacokinetics research [ D ]. Jiangsu: Jiangsu university, 2017.)
The long circulating liposome can be prepared by adding polyethylene glycol-derivatized phospholipid in a certain proportion into a common liposome prescription. The PEG long chain forms a steric hindrance and a hydrophilic protective layer on the surface of the liposome, so that the recognition and the uptake of phagocytes to the liposome can be prevented, the circulation time of the liposome in blood is prolonged, and the long circulation performance of the liposome is endowed. (Colloids and Surfaces B: Biointerfaces 155(2017):266-
The polymer nano micelle is a drug-carrying system developed in recent years for insoluble drugs, and has a core-shell structure, wherein the core is a hydrophobic part, and the shell is a hydrophilic part. The polymer nano micelle can load the insoluble drug at the nuclear shell boundary layer to achieve the solubilization of the insoluble drug. Compared with common solubilizing agents Tween-80 and polyoxyethyl castor oil, the polymer micelle carrier is usually made of biodegradable materials and has high safety, so the polymer micelle carrier has good application prospect as a carrier auxiliary material of insoluble drugs (Journal of Controlled Release,2001,73, 137-172; Pharmaceutical Research,2007,24(1), 1-16). At present, a plurality of nanometer preparations of taxane medicines are researched (CN103990135A, CN107625730A and CN104758256A), and the nanometer preparations solve the problems of poor water solubility, more adverse reactions, low bioavailability and the like of the taxane medicines to a certain extent. However, the existing taxane drug nano-preparations have the problems of low drug loading, low encapsulation efficiency and poor stability (CN 103990135A; CN 107625730A; CN 104758256A; CN 102293736A; CN 101732234A; CN 101804021A; and CN 101829046A). The drug loading rate of the existing nano preparation is generally lower than 5 percent, the encapsulation rate is less than 90 percent, so that a large amount of carrier materials can be introduced into the preparation, and the use of the nano preparation in clinic can be limited because many nano materials are not verified in clinic safety and even have the hidden danger of generating toxic substances by degradation in human bodies (Journal of Controlled Release,2009,133, 11-17; International Journal of pharmaceuticals, 2014,464(1-2): 178-); on the other hand, most of the nano preparations of the insoluble drugs have the problem of poor stability, the encapsulation rate is reduced in a short time, and the drug leakage is caused, particularly for the taxane drugs, when the encapsulation rate is reduced to be below 95%, insoluble particles appear in the liquid medicine, the quality of the preparation is influenced, and the nano preparations cannot be really applied in clinic (CN103990135A, CN107625730A, CN 104758256A); meanwhile, the preparation process of many nano preparations is very complicated before use, and some nano preparations even need a heating and stirring process, which brings many risks to clinical application (CN 101972480A; Drug Delivery and transportation Research,2018,5, 1365-.
Polyethylene glycol-derivatized phospholipids, such as PEG-DSPE, have a series of unique advantages as micelle drug delivery systems, and PEG-DSPE polymer molecules are Pharmaceutical excipients that have been approved by FDA, and have good safety (Journal of Controlled Release,2013,171, 133-. Our previous studies showed that some water-soluble small molecule drugs can be successfully loaded into the formed PEG-DSPE nano-micelles in an aqueous solution in a simple one-step self-assembly manner (Pharmaceutical Research,2010,27(2), 361-. Meanwhile, recently, the PEG-DSPE nano-micelle has the function of a natural molecular chaperone, a hydrophilic nano-cage formed by the shell of the PEG-DSPE nano-micelle can accommodate an A chain and a B chain of insulin, promote the correct folding of the two chains and prevent the aggregation of the insulin, and the PEG-DSPE nano-micelle can accommodate protein molecules with the molecular weight of less than 20KD according to the spatial structure of the formed nano-cage (Biomaterials,2016,77, 139-. Although PEG-DSPE has excellent drug loading capacity, it has a certain structural selectivity for The loaded drugs, such as doxorubicin hydrochloride, vinorelbine tartrate and vincristine sulfate, which can be successfully loaded into PEG-DSPE micelles with an entrapment rate of 99.9% (CN 1840193A; CN 101322681A; CN 1739525A; CN101138545A), however, PEG-DSPE micelles do not have The capacity of loading all small-molecule drugs, such as gemcitabine hydrochloride and paclitaxel, which cannot be successfully and stably loaded into PEG-DSPE micelles, and cannot achieve The purpose of clinical medication (Pharmaceutical Research,2010,27(2), 361-370; Pharmaceutical Transactions of The Royal Society A,20120309,371; Molecular Cancer Therapeutics,2014,13(12), 2864-2875).
Disclosure of Invention
The invention aims to solve the technical problem that the prior art can not effectively prepare the taxane medicine nano micelle preparation with high drug loading, high encapsulation rate and good stability; and provides a micelle containing taxane drugs, a preparation method and application thereof. Low drug loading, low encapsulation efficiency and poor stability are the main factors influencing the clinical application of the nano-drug preparation. The taxane medicine nano-micelle provided by the invention contains taxane medicines with effective treatment amount; the drug loading is as high as 6.25-9.09%, the encapsulation rate is 98-100%, and the stability of the re-dissolved nano micelle (and the encapsulation rate is 98-100%) is more than 24 hours; can effectively solve the problems in clinical application. Meanwhile, the taxane drug nano micelle provided by the invention has the characteristics of large drug-loading rate, high encapsulation rate and good stability, is a system with stable dynamics, is simple to prepare clinically, does not need a special infusion device, has a targeting effect in vivo, can increase the distribution of the drug in tumor tissues, improves the curative effect and reduces the toxicity.
The present invention solves the above-mentioned problems by the following technical means.
The invention provides a micelle containing a substance A, which comprises the following components in parts by weight: 1 part of the substance A and 5 to 25 parts of polyethylene glycol-derivatized phospholipid; the substance A is docetaxel and/or cabazitaxel; the polyethylene glycol-derivatized phospholipid comprises a polyethylene glycol part and a phospholipid part, wherein the phospholipid part is di (C)12-C24Fatty acyl) phosphatidylethanolamine.
In one embodiment of the present invention, the phospholipid moiety of the polyethylene glycol-derivatized phospholipid may be distearoyl phosphatidyl ethanolamine (C)18DSPE for short), phosphatidylethanolamine dipalmitate (C)16DPPE for short), dimyristoyl phosphatidyl ethanoneAlcohol amine (C)14DMPE for short) and dioleoylphosphatidylethanolamine (C)18DOPE for short); distearoyl phosphatidyl ethanolamine (DSPE) is preferred.
In the polyethylene glycol-derived phospholipid, the molecular weight of polyethylene glycol is 1000-5000, for example, the molecular weight of polyethylene glycol is 2000.
In a certain embodiment of the present invention, the polyethylene glycol-derivatized phospholipid may be polyethylene glycol 1000 distearoyl phosphatidyl ethanolamine (PEG1000-DSPE), polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine (PEG2000-DSPE), polyethylene glycol 5000 distearoyl phosphatidyl ethanolamine (PEG5000-DSPE), polyethylene glycol 2000 dioleoyl phosphatidyl ethanolamine (PEG2000-DOPE), polyethylene glycol 2000 dipalmitoyl phosphatidyl acetamide (PEG2000-DPPE), or polyethylene glycol 2000 dimyristoyl phosphatidyl ethanolamine (PEG 2000-DMPE); PEG2000-DSPE is preferred because PEG-DSPE polymer molecules are a pharmaceutically acceptable excipient that has been approved by the FDA and are highly safe.
In one scheme of the invention, the paint comprises the following components in parts by weight: 1 part of the substance A and 10 to 15 parts of polyethylene glycol-derivatized phospholipid.
In one embodiment of the present invention, the micelle of the substance a is composed of the substance a and the polyethylene glycol-derivatized phospholipid.
In one scheme of the invention, the paint comprises the following components in parts by weight: 1 part docetaxel and 5 parts or 15 parts polyethylene glycol derivatized phospholipid (e.g., PEG 2000-DSPE). For example, it consists of 1 part docetaxel and 5 parts or 15 parts of the polyethylene glycol-derivatized phospholipid (e.g., PEG 2000-DSPE). The drug loading can be as high as 6.25%.
In one scheme of the invention, the paint comprises the following components in parts by weight: 1 part of cabazitaxel and 5 parts, 10 parts or 15 parts of polyethylene glycol-derivatized phospholipid (e.g., PEG1000-DSPE, PEG2000-DSPE, PEG5000-DSPE, PEG2000-DOPE, PEG2000-DPPE, PEG 2000-DMPE). For example consisting of 1 part cabazitaxel and 5, 10 or 15 parts polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine, or consisting of 1 part cabazitaxel and 10 parts polyethylene glycol 1000 distearoyl phosphatidyl ethanolamine, polyethylene glycol 5000 distearoyl phosphatidyl ethanolamine, polyethylene glycol 2000 dioleoyl phosphatidyl ethanolamine, polyethylene glycol 2000 dipalmitoyl phosphatidyl acetamide or polyethylene glycol 2000 dimyristoyl phosphatidyl ethanolamine). The drug loading rate can reach 9.09%.
In the invention, the micelle containing the substance A is in a spherical shape with a core-shell structure; wherein, the inner core is a phospholipid layer, the outer shell is a polyethylene glycol (PEG) layer, and the substance A is distributed in the polyethylene glycol derivatization phospholipid micelle (namely, the substance A is wrapped at the nuclear shell boundary layer inside the micelle).
In one embodiment of the invention, the size of the micelles may be in the range of 5 to 50nm, preferably 10 to 20 nm.
In one embodiment of the present invention, the micelle has a Polydispersity (PDI) of less than 0.3 and a uniform particle size distribution; preferably less than 0.2, for example, when said substance a is cabazitaxel, the Polydispersity (PDI) is between 0.100 and 0.140. When the substance A is docetaxel, the polydispersity is 0.100-0.180.
In one aspect of the invention, when the polyethylene glycol-derivatized phospholipid is PEG2000-DSPE, the average diameter of the micelles is 10-15nm (e.g., 12 nm).
In one embodiment of the invention, when the polyethylene glycol-derivatized phospholipid is PEG2000-DSPE, the aggregation number of the micelle is about 90.
Another object of the present invention is to provide a method for preparing a taxane-containing material, which comprises the following steps:
step 1: removing the organic solvent from the solution to obtain a polymer lipid membrane containing taxane drugs; wherein, the solution consists of 1 part of taxane drugs, 5 to 25 parts of polyethylene glycol-derivatized phospholipid and the organic solvent; the taxane drugs are docetaxel and/or cabazitaxel; the polyethylene glycol-derivatized phospholipid comprises a polyethylene glycol part and a phospholipid part, wherein the phospholipid part is di (C)12-C24Fatty acyl) phosphatidylethanolamine;
step 2: and (3) hydrating the polymer lipid membrane obtained in the step (1) in an aqueous solvent to obtain a substance containing the taxane drugs.
In one embodiment of the present invention, the phospholipid moiety in the polyethylene glycol-derivatized phospholipid may be one or more of distearoyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), and dioleoyl phosphatidyl ethanolamine (DOPE); distearoyl phosphatidyl ethanolamine (DSPE) is preferred.
In the polyethylene glycol-derived phospholipid, the molecular weight of polyethylene glycol is 1000-5000, for example, the molecular weight of polyethylene glycol is 2000.
In a certain embodiment of the present invention, the polyethylene glycol-derivatized phospholipid may be polyethylene glycol 1000 distearoyl phosphatidyl ethanolamine (PEG1000-DSPE), polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine (PEG2000-DSPE), polyethylene glycol 5000 distearoyl phosphatidyl ethanolamine (PEG5000-DSPE), polyethylene glycol 2000 dioleoyl phosphatidyl ethanolamine (PEG2000-DOPE), polyethylene glycol 2000 dipalmitoyl phosphatidyl acetamide (PEG2000-DPPE), or polyethylene glycol 2000 dimyristoyl phosphatidyl ethanolamine (PEG 2000-DMPE); PEG2000-DSPE is preferred because PEG-DSPE polymer molecules are a pharmaceutically acceptable excipient that has been approved by the FDA and are highly safe.
In one scheme of the invention, the solution comprises the following components in parts by weight: 1 part of taxane medicine and 10 to 15 parts of polyethylene glycol-derivatized phospholipid.
In one scheme of the invention, the solution comprises the following components in parts by weight: 1 part docetaxel and 5 parts or 15 parts polyethylene glycol derivatized phospholipid (e.g., PEG 2000-DSPE).
In one scheme of the invention, the solution comprises the following components in parts by weight: 1 part cabazitaxel and 5, 10 or 15 parts polyethylene glycol derivatized phospholipid (e.g., consisting of 1 part cabazitaxel and 5, 10 or 15 parts polyethylene glycol 2000 distearoylphosphatidylethanolamine, or, alternatively, consisting of 1 part cabazitaxel and 10 parts polyethylene glycol 1000 distearoylphosphatidylethanolamine, polyethylene glycol 5000 distearoylphosphatidylethanolamine, polyethylene glycol 2000 dioleoylphosphatidylethanolamine, polyethylene glycol 2000 dipalmitoylphosphatidylacetamide acetamide or polyethylene glycol 2000 dimyristoylphosphatidylethanolamine).
Instep 1, the organic solvent may be one or a mixture of at least two of an organic solvent conventional in such production methods in the art, such as an alcohol solvent (again, for example, methanol and/or ethanol), a halogenated hydrocarbon solvent (again, for example, chloroform) and a nitrile solvent (again, for example, acetonitrile), preferably ethanol and/or acetonitrile, most preferably ethanol.
It will be understood by those skilled in the art that the solution of the taxane drug, the polyethylene glycol-derivatized phospholipid and the organic solvent in step 1) is to dissolve the drug and the polyethylene glycol-derivatized phospholipid in the organic solvent, so that the two can be contacted at the molecular level, and therefore, the amount of the organic solvent is not particularly limited, and can be controlled to dissolve the two.
In the present invention, the operation of removing the organic solvent may be a conventional operation in the art, for example, the organic solvent is removed by a reduced pressure rotary evaporation method and/or the organic solvent is removed under vacuum; in the invention, the rotary evaporation temperature is 30-60 ℃, preferably 40 +/-5 ℃, and the rotation speed is 10-500 r/min, preferably 60-150 r/min.
In the present invention, the aqueous solvent is selected from one or a mixture of at least two of deionized water, a PBS buffer solution (e.g., 0.01M-0.2M phosphate buffered saline), physiological saline (0.9% aqueous sodium chloride), glucose injection (e.g., 5-50g/mL), and amino acid injection (e.g., 5-10g/mL), preferably deionized water.
In the present invention, the mass-to-volume ratio of the taxane in the aqueous solvent may be 0.5mg/mL to 15mg/mL, preferably 5mg/mL to 10mg/mL (for example, 5mg/mL to 6mg/mL when the taxane is docetaxel, and 5mg/mL to 8mg/mL when the taxane is cabazitaxel).
The hydration operation can be the conventional operation of the hydration in the field, and in the invention, the hydration operation can be vortex shaking or ultrasonic hydration. The vortex shaking hydration can be 1-3 hours. The time of ultrasonic hydration can1-60 minutes, preferably 10-30 minutes (such as 20 minutes), and the ultrasonic sound intensity can be 0.1-15 w/cm2(e.g., 10 w/cm)2) The frequency is 0.1 to 100 kHz. (e.g., 40kHz)
The temperature of the hydration may be a temperature conventional in such hydration operations in the art, for example, from 0 ℃ to 60 ℃; in the present invention, a water bath of 20 ℃ to 40 ℃ (e.g., 20 ℃ to 40 ℃); for example 30-40 deg.c.
In one embodiment of the present invention, the hydration may be performed without a pharmaceutical excipient or in the presence of a pharmaceutical excipient. Preferably, it is carried out in the absence of a pharmaceutical excipient.
In a certain embodiment of the present invention, in the preparation method, thestep 2 may further include a filtration step, and the filtration step may be a filtration step conventional in the methods in the art; for example, it is the following steps: after hydration (for example, when the aqueous solvent is deionized water), performing extrusion filtration (to remove impurities) through a nano-scale filter membrane to obtain the substance containing the taxane drugs; or, after hydration (for example, when the aqueous solvent is deionized water), the mixture with the pharmaceutical excipients is extruded and filtered (to remove impurities) through a nano-scale filter membrane to obtain the substance containing the taxane drugs; preferably, after the mixture is hydrated in deionized water, the mixture with the pharmaceutical excipients is extruded and filtered (impurities are removed) through a nano-scale filter membrane to obtain the substance containing the taxane drugs.
Instep 2, the pharmaceutical excipient may be a pharmaceutical excipient conventional in such micelles in the art, such as a lyophilized excipient (or lyoprotectant). The freeze-drying excipient can be one or more of glucose, mannitol, lactose, trehalose, sucrose, dextran, glycine, fructose and sorbitol, and is preferably glucose. The mass ratio of the lyophilized excipient to the taxane may be 0 to 25:1 (e.g., 15.6:1, 20.8: 1). The freeze-drying excipient has another function, can help freeze-drying powder to be quickly redissolved, and reduces the time required for redissolving the freeze-drying powder to be clear.
In the invention, the nano-scale filter membrane can be selected from filter membranes with the pore diameter of 10-300 nm, preferably filter membranes with the pore diameter of 220nm, and the filter membrane material is a mixed cellulose ester microporous filter membrane, a nylon filter membrane, a polytetrafluoroethylene filter membrane, a polyvinylidene fluoride membrane, a polyether sulfone filter membrane or a polypropylene filter membrane, preferably a polyvinylidene fluoride membrane or a polyether sulfone filter membrane, and most preferably a polyether sulfone filter membrane.
In a certain aspect of the invention, it may comprise the steps of: dissolving the taxane drugs and the polyethylene glycol-derivatized phospholipid in the organic solvent (such as ethanol) in parts by weight, performing rotary evaporation (such as at 30-60 ℃) to remove the organic solvent to form a uniform polymer lipid membrane, adding an aqueous solvent (such as deionized water), performing ultrasonic hydration in a water bath at 20-40 ℃ (such as at 30-40 ℃), and filtering with a nano-scale filter membrane (such as 220nm) to remove impurities to obtain the material containing the taxane drugs; or mixing with pharmaceutical adjuvants after hydration, and filtering with nanometer filter membrane (such as 220nm) to remove impurities to obtain the substance containing taxane.
In one embodiment of the present invention, the taxane-containing material is composed of the taxane and the polyethylene glycol-derivatized phospholipid, or further comprises the aqueous solvent; or, when the pharmaceutic adjuvant exists, the substance containing the taxane medicines is composed of the taxane medicines, the polyethylene glycol-derivatized phospholipid and the pharmaceutic adjuvant, or further comprises the aqueous solvent.
In one embodiment of the present invention, the preparation method further comprises a lyophilization step, and the operation of the lyophilization step can be a routine operation in the art.
In one embodiment of the present invention, the substance containing taxane is a micelle containing the taxane; for example, micelles containing substance a as described above; it is obtained by the following principle: the polyethylene glycol-derivatized phospholipid in the polymer lipid membrane containing the taxane drugs is self-assembled in the aqueous solvent as a carrier material through interaction force to form a composite skeleton through hydration, and the taxane drugs are wrapped in micelles formed by the polyethylene glycol-derivatized phospholipid.
In the invention, when the substance containing the taxane medicines is micelles containing the taxane medicines, the particle size and the particle size distribution of the micelles can be reduced by the processes of high-pressure homogenization, high-speed homogenization or ultrasound and the like. The size of the micelles may range from 5 to 50nm, preferably from 10 to 20 nm. The Polydispersity (PDI) of the micelle is less than 0.3, and the particle size distribution is relatively uniform; preferably less than 0.2, for example, when said substance a is cabazitaxel, the Polydispersity (PDI) is between 0.100 and 0.140. When the substance A is docetaxel, the polydispersity is 0.100-0.180. The average diameter of the micelles may be 10 to 15 nm. When the polyethylene glycol-derivatized phospholipid is PEG2000-DSPE, the aggregation number of the micelle can be 90. The encapsulation rate of the taxane medicine can be more than 98% (such as 100%). Wherein the room temperature stability of the docetaxel nano-micelle is not shorter than 4 hours. Wherein the room temperature stability of the cabazitaxel nano-micelle is not shorter than 8 hours.
The invention also provides a substance containing the taxane medicines, which is prepared according to the preparation method.
It will be understood by those skilled in the art that the taxane-containing material is "substantially free" of organic solvents; as used herein, "substantially free" means no or insignificant amount (i.e., an amount that has no measurable effect on the physical properties of the taxane-containing material). For example, an organic solvent may not be included, or an organic solvent as described above may also be included; the organic solvent is an organic solvent (such as methanol, ethanol, chloroform, acetonitrile or a mixture thereof, such as ethanol and/or acetonitrile, and ethanol) which is remained in the preparation process and is conventional in the preparation method in the field. The organic solvent may be within 2% (weight percent organic solvent/drug).
The invention provides a pharmaceutical composition, which comprises the micelle containing the substance A or the substance containing the taxane medicaments and pharmaceutic adjuvants.
The pharmaceutical composition can be a freeze-dried preparation, an injection, an oral preparation and the like, and is prepared by the conventional preparation process in the field, such as freeze drying, spray drying, reduced pressure evaporation and the like.
In one embodiment of the present invention, the pharmaceutical composition may be in the form of a solution or a lyophilized form, preferably a lyophilized form, as required.
The pharmaceutic adjuvant can be a conventional pharmaceutic adjuvant in the pharmaceutical composition in the field; for example, an aqueous solvent and/or a lyophilization excipient; the aqueous solvent can be one or a mixture of at least two of deionized water, PBS buffer solution (such as 0.01M-0.2M phosphate buffer solution), physiological saline (0.9% sodium chloride aqueous solution), glucose injection (such as 5-50g/mL) and amino acid injection (such as 5-10g/mL), and is preferably deionized water; the freeze-drying excipient can be one or more of glucose, mannitol, lactose, trehalose, sucrose, dextran, glycine, fructose and sorbitol, and is preferably glucose.
The invention also provides an application of the micelle containing the substance A or the substance containing the taxane medicines in the preparation of medicines for treating cancers. Wherein said substance A or said taxane is present in a therapeutically effective amount. The cancer is selected from breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.
For a better understanding of the present invention, some terms of art are explained below.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
In the present application, a "pharmaceutical composition" refers to a formulation of a compound of the present invention with a vehicle generally accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutical excipient. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of active ingredients and exert biological activity.
The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are primarily used to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents and sweeteners.
The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.
The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration.
The term "micelle" refers to a molecule that is capable of spontaneously polymerizing to form a micelle when the concentration of amphiphilic molecules in an aqueous solution exceeds a Critical Micelle Concentration (CMC). Micelles, unlike liposomes, do not have the structural features of lipid bilayers. Generally, micelles have a structure in which the hydrophobic portion faces inward, forming a hydrophobic core, and the hydrophilic portion faces outward, forming a hydrophilic surface. The micelle has small particle size, and the average particle size is about 10-50 nm. It is therefore not only a thermodynamically stable system, but also a kinetically stable system. In addition, the micelle particles are not easy to aggregate and stratify, and the entrapment capacity is high, namely, higher drug amount can be entrapped at low concentration.
The term "micelle aggregation number" is a measure of the size of the micelle, i.e., the number of surfactant molecules or ionic monomers associated into the micelle.
The term "therapeutically effective amount" refers to the amount of substance a or the taxane that produces a therapeutic effect. According to the invention, the unit dose (amount of drug used per square meter of body surface area) of the substance A or the taxane is 5-100 mg/m2Preferably, the unit dosage is 10-20 mg/m2The optimal unit dose is 20mg/m2The dosage will be adjusted to the needs of each particular individual.
In the invention, the polyethylene glycol-derivatized phospholipid is formed by combining polyethylene glycol molecules with nitrogenous bases on phospholipid molecules through covalent bonds. In the structure, the fatty acid of the phospholipid part contains 10 to 24 carbon atoms, preferably 12, 14, 16, 18, 20, 22 and 24 carbon atoms (for example, 14, 16 and 18 carbon atoms), and more preferably 18 carbon atoms; the fatty acid chain may be saturated or partially saturated, and preferably, the fatty acid is one or more of lauric acid, myristic acid, palmitic acid, stearic acid or oleic acid or linoleic acid, eicosane fatty acid, behenic acid and lignocelate.
The micelle preparation takes polyethylene glycol-derivatized phospholipid as a main matrix, and polyethylene glycol molecules form a hydrophilic protective layer outside a hydrophobic core for encapsulating the medicament, so that the contact between the medicament and protein molecules such as enzyme in blood can be avoided, the nano-micelle is protected from being phagocytized by a reticuloendothelial system in vivo, and the retention time of the nano-micelle in blood circulation is prolonged; the medicine is encapsulated in the hydrophobic core in the micelle, so that the medicine can be prevented from being damaged by external factors (water, oxygen and light), and the stability of the medicine in the storage process is greatly improved; in addition, the dynamic property of the distribution of the medicine in the body is changed, so that the curative effect is enhanced, and the toxicity is reduced.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: 1) the provided taxane medicine micelle preparation selects a safe polymer carrier material with simple components, improves the solubility of the taxane medicines and the compliance of the preparation, realizes the medicine loading capacity with sufficient clinical effect, has the medicine loading rate as high as 6.25-9.09 percent and the medicine encapsulation rate as high as 98-100 percent, and can realize the loading of the taxane medicines under the condition of lower auxiliary material dosage.
2) The taxane medicine nano powdery preparation improves the stability, compliance and safety of the medicine. The powdery preparation can be hydrated into a nano micelle preparation after being dissolved, has good stability, has the stability (the encapsulation rate is 98-100%) of being placed at room temperature for more than 24 hours, can be directly used for intravenous injection, and does not need a special infusion device. Under the in vitro condition, the stability is higher because the competitive combination of albumin, immunoglobulin, hemoglobin and other in vivo related proteins is not available; after the drug-loaded micelle enters blood, the micelle can gradually dissociate to release drug molecules due to the interaction of blood flow velocity, blood pressure and protein.
3) The average particle size of the taxane micelle is 10-20nm, the passive targeting of the nano preparation to the tumor can be realized by utilizing the permeation enhancement and retention effect (EPR effect) of the tumor tissue, the selective distribution of the medicine in the tumor tissue is promoted, the medicine effect can be increased, and the toxic and side effects of a system can be reduced.
4) The preparation process is simple, has good repeatability and is suitable for industrial production.
Drawings
FIG. 1 is a graph of the effect of different cabazitaxel formulations on mouse body weight in example 9.
FIG. 2 is a graph showing the cytotoxic effect of cabazitaxel micelles on MC-38 cells in example 10.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Particle size analysis of drug loaded micelles
The particle size distribution of the drug-loaded micelles was determined by Dynamic Light Scattering (DLS). The sample is diluted to 1mg/mL by physiological saline, 1mL of the sample is taken, and the particle size distribution and the polydispersity PDI of the drug-loaded micelle are measured by a Malvern Zetasizer Nano-ZS light scattering particle size analyzer. The wavelength of the laser beam of the instrument is set to 633nm, and the included angle between the incident light and the scattered light beam is 90 degrees. For each sample, 20 cycle times were measured, and the measurement temperature was set to 25 ℃. The assay results for each formulation are the average of the assay results for 3 batches of formulation. The Polydispersity (PDI) indicates the degree of uniformity of particle size, with particles being more uniform as the polydispersity decreases.
Drug encapsulation efficiency and drug loading
1mL of the drug-loaded micelle solution is taken and filtered by a 220nm microporous filter membrane, the drug-loaded micelle and the concentration of the drug-loaded micelle in the filtrate are measured by adopting a high performance liquid chromatography, and the entrapment rate and the drug-loaded amount are calculated by adopting the following formula.
The encapsulation efficiency is C2/C1 × 100%
Wherein, C1: drug concentration in the drug-loaded micelle solution; c2: drug concentration in the drug-loaded micellar solution after filtration through a 220nm filter (the drug not encapsulated by the micelles is removed by filtration in solid form). The concentration of the drug released from the drug-loaded micelles in the solution was determined by high performance liquid chromatography, respectively.
The drug loading rate is (C2 XV)/(C1 XV + M). times.100%
In the formula, V: the volume of the drug-loaded micelle; m: input amount of carrier in drug-loaded micelle
Determination of the number of accumulations based on static light scattering methods
An eighteen-angle Static Light Scattering (SLS) was used in conjunction with a differential detector to measure the molar mass Mw of the micelles. After toluene calibration, 5 samples with different concentrations were injected into the detector from low to high in sequence, the injection volume was 2ml each time, the flow rate was 0.5ml/min, and the detection temperature was 25 ℃. After data collection, analysis was performed using the ASTRA software, and the aggregation number was calculated according to the following formula.
Aggregation number Mw/2805
Reference pharmacopoeia method for detecting residual solvent
Example 1 preparation of docetaxel nanomicelle formulation and stability study
The prescription is shown in table 1:
table 1 example 1 docetaxel nanomicelle formulation
| Medicine | Lipid/medicine (mass ratio) | Drug concentration (mg/mL) | Hydration solvent |
| Docetaxel | 15:1 | 6 | Deionized water |
| Docetaxel | 5:1 | 6 | Deionized water |
The preparation process comprises the following steps: weighing docetaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine (PEG2000-DSPE, purchased from Avanti company) according to the above formula proportion, placing into a rotary evaporation bottle, adding appropriate amount of ethanol, gently shaking to completely dissolve the medicine and lipid to obtain clear transparent solution, pumping off the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at a rotation speed of 90r/min and a water bath temperature of 40 ℃, uniformly distributing the lipid and the medicine on the inner wall of the rotary evaporation bottle after the organic solvent is pumped off, adding deionized water after the rotary evaporation bottle is cooled to room temperature, performing ultrasonic treatment in a water bath at 30 ℃ for 5min (40KHz,50 w; the same below) to completely dissolve a lipid membrane (medicine concentration of 6mg/mL), adding appropriate amount of freeze-drying excipient glucose to make the final glucose concentration of 125mg/mL, gently shaking to completely dissolve the glucose, filtering with a microporous membrane of 220nm, subpackaging and freeze-drying to obtain the docetaxel nano micelle preparation freeze-dried powder with large drug-loading rate, high encapsulation rate and high stability.
The molar mass of the polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine empty micelle is about 2.52x 105gmol-1The aggregation number is about 90. After loading the drug, the molar mass of the micelle increased with the increase of the drug loading, but the aggregation number did not change significantly, and was still about 90.
The obtained sample is a clear and transparent solution after redissolving, the particle size distribution is between 5nm and 20nm, the average particle size is 12nm, and the PDI is 0.107; the drug loading is 6.25 percent and 16.67 percent (mass percentage), the encapsulation rate is 98 percent to 100 percent, and the stability (encapsulation rate is 98 percent to 100 percent) of the drug placed at 25 ℃ is more than 4 hours (wherein, when the ratio is 15:1, the time is more than 24 hours). (the aggregation number did not change significantly and remained 90).
Example 2: preparation and stability investigation of cabazitaxel nano micelle preparation
The prescription is shown in table 2:
table 2 example 2 cabazitaxel nanomicelle formulation
| Medicine | Lipid/medicine (mass ratio) | Medicine concentrateDegree (mg/mL) | Hydration solvent |
| Cabazitaxel | 10:1 | 8 | Deionized water |
The preparation process comprises the following steps: weighing cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the formula, placing the cabazitaxel and the polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, drying the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at the rotating speed of 90r/min and the water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is dried, cooling the rotary evaporation bottle to room temperature, adding deionized water, carrying out ultrasonic treatment in a water bath at 30 ℃ for 20min to completely dissolve a lipid membrane, adding a proper amount of a freeze-drying excipient glucose to ensure that the final concentration of the glucose is 125mg/mL, gently shaking to completely dissolve the glucose, filtering by a 220nm microporous filter membrane, and carrying out split charging and freeze drying to obtain the docetaxel nano-micelle freeze-dried powder with large drug loading rate, high encapsulation efficiency and high stability. The obtained sample is a clear and transparent solution after redissolving, the particle size distribution is between 5 and 20nm, the average particle size is 12nm, and the PDI is 0.114; the drug loading is 9.1 percent, the encapsulation rate is 98 to 100 percent, and the stability (the encapsulation rate is 98 to 100 percent) of the drug placed at 25 ℃ is more than 24 hours. (the aggregation number does not change significantly, and is still about 90.)
Example 3: the paclitaxel nano micelle has high encapsulation rate, but has poor stability and cannot meet the clinical medication requirement
The prescription is shown in table 3:
table 3 example 3 paclitaxel nanomicelle formulation
| Medicine | Lipid/medicine (mass ratio) | Drug concentration (mg/mL) | Hydration solvent |
| Paclitaxel | 15:1 | 5 | Deionized water |
The preparation process comprises the following steps: weighing paclitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the formula, placing into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and lipids to obtain a clear transparent solution, pumping the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at a rotating speed of 90r/min and a water bath temperature of 40 ℃, pumping the organic solvent, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle, cooling the rotary evaporation bottle to room temperature, adding deionized water, performing ultrasonic treatment in a water bath at 30 ℃ for 20min to completely dissolve a lipid membrane, filtering with a 220nm microporous membrane (or adding a proper amount of freeze-drying excipient glucose to ensure that the final concentration of the glucose is 125mg/ml, gently shaking to completely dissolve the glucose, filtering with a 220nm microporous membrane), ensuring that the encapsulation rate is 99%, then placing the rotary evaporation bottle to room temperature to examine the stability of the rotary evaporation bottle, the paclitaxel nano micelle preparation has a turbidity tendency after being placed for 10min, and has an increased turbidity and a precipitate separation after 20min, which indicates that the paclitaxel micelle is very unstable when being placed at room temperature and cannot meet clinical medication.
Example 4: influence of freeze-drying excipient on reconstitution time and room-temperature placement stability of cabazitaxel nano micelle freeze-drying powder
The prescription is shown in table 4:
table 4 example 4 cabazitaxel nanomicelle formulation
| Medicine | Lipid/medicine (mass ratio) | Drug concentration (mg/mL) | Hydration solvent |
| Cabazitaxel | 10:1 | 8 | Deionized water |
The preparation process comprises the following steps: weighing cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the formula, placing the cabazitaxel and the polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, drying the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at the rotating speed of 90r/min and the water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is dried, cooling the rotary evaporation bottle to the room temperature, adding deionized water, carrying out ultrasonic treatment in the water bath at the temperature of 30 ℃ for 20min to completely dissolve a lipid membrane, adding a proper amount of freeze-drying excipient (glucose, mannitol, lactose or trehalose) to ensure that the final concentration of the freeze-drying excipient is 125mg/mL, gently shaking to completely dissolve the freeze-drying excipient, filtering by a microporous filter membrane of 220nm, and carrying out freeze-drying. Taking each bottle of freeze-dried powder containing different freeze-drying excipients, redissolving the freeze-dried powder by using water for injection, and recording the time required by redissolving the cabazitaxel micelle preparation to be clear and the clarity and the encapsulation rate of the cabazitaxel micelle preparation after the cabazitaxel micelle preparation is placed at room temperature (25 ℃) for 24 hours. The results are shown in Table 5. The test result shows that the addition of the freeze-drying excipient does not influence the stability of the preparation, but the appropriate freeze-drying excipient can obviously reduce the time required for re-dissolving the freeze-drying powder to be completely dissolved, and in consideration of the convenience of clinical use, glucose is selected as the freeze-drying excipient of the taxane micelle preparation!
TABLE 5 time required for reconstitution and stability study of cabazitaxel nanomicelle formulations containing different freeze-drying excipients
Stability greater than 100% is a normal error range due to measurement errors.
EXAMPLE 5 Cabazitaxel Nanosplex formulations and Cabazitaxel injections (commercially available) comparison of stability at 37 deg.C
The cabazitaxel nano-micelle preparation and the cabazitaxel injection in the example 2 are redissolved or diluted by water for injection until the final concentration of the medicine is 5mg/mL, then the two preparations are placed in a 37 ℃ incubator, the clarity of the preparations is observed at different time points, and after the two preparations are placed for 4 hours, the medicine crystal form in a cabazitaxel injection (sold in the market) solution is found, and the medicine crystal is increased and a large amount of precipitate is generated at the bottom after 8 hours, while the cabazitaxel nano-micelle preparation is still a clear and transparent liquid, and the encapsulation rate is 99%, which indicates that the stability of the cabazitaxel nano-micelle preparation is far better than that of the cabazitaxel injection (sold in the market).
Example 6 lipid to drug ratio (weight ratio) affects the stability of taxane drug nanomicelle formulations
See table 6 for prescription:
table 6 example 6 cabazitaxel nanomicelle formulation
| Medicine | Lipid/medicine (mass ratio) | Drug concentration (mg/mL) | Hydration solvent |
| Cabazitaxel | 5:1 | 5 | Deionized water |
| Cabazitaxel | 10:1 | 5 | Deionized water |
| Cabazitaxel | 15:1 | 5 | Deionized water |
The preparation process comprises the following steps: weighing cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the formula, placing the cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, drying the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at the rotating speed of 90r/min and the water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is dried, cooling the rotary evaporation bottle to room temperature, adding deionized water, performing ultrasonic treatment in a water bath at 30 ℃ for 20min to completely dissolve a lipid membrane, filtering by using a 220nm microporous filter membrane, placing cabazitaxel nano micelle preparations with different drug-lipid ratios at room temperature (25 ℃), observing the stability of the change investigation period of the preparation clarity, and obtaining the results shown in Table 7.
Table 7 ratio of lipid to drug effects on stability of cabazitaxel nanomicelle formulations
When the same dose of drug is administered to a patient, the lower the drug loading of the nano-formulation, the more carrier is present in the patient and the greater the potential toxic side effects.
When the freeze-dried form is 5:1, the mixture is redissolved by water for injection; after standing at 25 ℃ for 2 hours, the solution had a clarity of a clear and transparent solution, an encapsulation efficiency (%) of 99.1%, a particle diameter (nm) of 12.1 and a PDI of 0.107. Therefore, the solution was prepared in the form of a desiredsolution 2 hours before use.
Example 7 Effect of different hydration temperatures on Room temperature stability of Cabazitaxel Nanomangle formulations
See table 8 for prescription:
table 8 example 7 cabazitaxel nanomicelle formulation
The preparation process comprises the following steps: weighing cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the formula, placing the cabazitaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, drying the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at the rotating speed of 90r/min and the water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is dried, then adding deionized water after the rotary evaporation bottle is cooled to the room temperature, placing the rotary evaporation bottle in water baths at different temperatures according to the formula, carrying out ultrasonic treatment for 20min to completely dissolve a lipid membrane, filtering by using a microporous filter membrane of 220nm, placing cabazitaxel nano-micelle preparations prepared at different hydration temperatures to the room temperature (25 ℃), observing the stability of the change investigation period of the preparation clarity, and obtaining the results shown in Table 9.
TABLE 9 influence of hydration temperature on stability of cabazitaxel nanomicelle formulations at room temperature
NA denotes no measurement
Example 8 Polymer micelles formed by other kinds of polymer materials and taxanes have poor stability and cannot meet the clinical medication requirements
Prescription is shown in table 10:
TABLE 10 example 8 other classes of polymer nanomicelle formulations
The process flow comprises the following steps: weighing Cabazitaxel (CTX) and polymer materials according to the formula, placing the Cabazitaxel (CTX) and the polymer materials into a rotary evaporation bottle, adding a proper amount of chloroform, gently shaking to dissolve the CTX and the polymer materials to obtain a clear transparent solution, pumping the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporation drier at a rotating speed of 90r/min and a water bath temperature of 40 ℃, and uniformly distributing the polymer materials and the CTX on the inner wall of the rotary evaporation bottle after the organic solvent is pumped. Then, after the rotary evaporation bottle was cooled to room temperature, 10mL of deionized water was added, followed by sonication at 30 ℃ to completely dissolve the lipid membrane, and then it was left to room temperature (25 ℃) to examine its stability, with the results shown in Table 11. The test results suggest that although other polymer materials can also load taxane drugs, the polymer materials have the characteristic of poor stability and cannot meet the clinical application, so the polymer materials also have no application value.
TABLE 11 poor stability of polymer micelles formed by other kinds of polymer materials and taxanes
PLGA is poly (lactic-co-glycolic acid), PDLLA is racemic polylactic acid, PCL is polycaprolactone, and F127 is poloxamer.
Example 9
Effect of Cabazitaxel micelles on animal body weight
BALB/c mice (SPF grade), 6-8 weeks old, and 19-21g in body weight were divided into seven groups of 10 mice each, and the seven groups were treated with the following cabazitaxel micelles (M-CTX) (example 2) or cabazitaxel injection (CTX-Tween80), respectively: 12.5, 25 and 50mg/kg cabazitaxel micelles or cabazitaxel injections were administered once in the tail vein and the effect of the drug on the body weight of the mice was recorded. The results are shown in fig. 1, and it can be seen from the body weight results of the mice after administration that the toxicity of the cabazitaxel micelle preparation at the same dose is obviously lower than that of the cabazitaxel injection.
TABLE 12 Effect of different Cabazitaxel formulations on mouse body weight
Example 10
In vitro cell assay
Cytotoxicity of cabazitaxel micelles (example 2) and cabazitaxel injection (in-house) (CTX-Tween80) on MC-38 cultured in vitro was determined using MTT (dimethylthiazole blue) staining. In vitro cultured MC-38 cells (culture medium RPMI 1640 containing 10% fetal calf serum, penicillin and streptomycin double antibody, culture environment 37 deg.C, saturation humidity, andcarbon dioxide concentration 5%) are washed twice with PBS, trypsinized, counted by cell counting plate, and diluted to 2 × 104Cells were plated per ml in 96-well cell culture plates, 100. mu.L (2X 10) per well3Individual cells); culturing in normal culture medium overnight; adding a predetermined complete culture medium containing cabazitaxel micelles with different concentrations and cabazitaxel injection into each hole, continuously culturing six parallel samples with each concentration for 72 hours; PBS is washed to remove the culture medium containing the drug, 100 mul MTT solution is added into each hole, and the incubation is carried out for 4 hours at 37 ℃; discard MTT, add 100 μ L of dimethyl sulfoxide (DMSO) per well, shake gently for 10 minutes at room temperature to dissolve the purple crystals completely; the absorbance of the solution was measured at a wavelength of 570nm with a full-automatic enzyme calibration apparatus (Thermo) to plot cell growth curves at different drug concentrations (FIG. 2).
TABLE 13 cytotoxic Effect of Cabazitaxel micelles on MC-38 cells
Experimental results show that the cabazitaxel micelle (M-CTX) has stronger growth inhibition effect on MC-38 cells than cabazitaxel injection (CTX-Tween80) under the same concentration, and the cabazitaxel micelle (M-CTX) has stronger killing effect on tumor cells than cabazitaxel injection (CTX-Tween 80).
Example 11: influence of freeze-drying excipient on re-dissolving time and room-temperature standing stability of docetaxel nano micelle freeze-drying powder
The prescription is shown in the table:
docetaxel nano micelle preparation prescription
The preparation process comprises the following steps: weighing docetaxel and polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine according to the proportion of the prescription, placing the docetaxel and the polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, pumping the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at a rotating speed of 90r/min and a water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is pumped, then adding deionized water after the rotary evaporation bottle is cooled to room temperature, carrying out ultrasonic treatment in a water bath at 30 ℃ for 20min to completely dissolve a lipid membrane, adding a proper amount of freeze-drying excipient (glucose, mannitol, lactose or trehalose) to ensure that the final concentration of the freeze-drying excipient is 125mg/mL, gently shaking to completely dissolve the freeze-drying excipient, then filtering by a microporous filter membrane of 220nm, and carrying out split charging and freeze-drying. Taking a bottle of freeze-dried powder containing different freeze-drying excipients, re-dissolving the freeze-dried powder with water for injection, and recording the time required by re-dissolving the docetaxel micelle preparation to be clear and the clarity and encapsulation rate of the docetaxel micelle preparation after the docetaxel micelle preparation is placed at room temperature (25 ℃) for 6 hours. The results are shown in the table. The test results show that the addition of the freeze-drying excipient does not affect the stability of the preparation, but the appropriate freeze-drying excipient can obviously reduce the time required for the freeze-drying powder to be redissolved to be completely dissolved!
Time required for redissolving and stability investigation of nano micelle preparation containing different freeze-drying excipients docetaxel
Stability greater than 100% is a normal error range due to measurement errors.
Example 12: pharmacodynamic study of docetaxel nano-micelle on melanoma tumor model
Pharmacodynamic experiments in a C57 mouse subcutaneous graft tumor model of human melanoma B16F10 were performed using prepared docetaxel nanomicelles (example 1): 20 qualified C57 tumorigenic animals of B16F10 were screened, randomly divided into 2 groups of 10 animals each, and administered with a common docetaxel injection (10mg/kg), docetaxel nanomicelle (10mg/kg), tail vein injection once every three days, three times in total. General clinical symptoms were observed 2 times a day in animals and tumor volume measurements were taken every 3 days (when tumor volume exceeded 5000mm3No longer recorded).
| Time (days)/tumor volume (mm)3) | DCX-TW80 | M- |
| 1 | 268.8 | 239.1 |
| 4 | 745.2 | 368.7 |
| 7 | 1645.4 | 627.6 |
| 10 | 3132.8 | 1115.1 |
| 13 | - | 1494.7 |
| 16 | - | 2623.8 |
| 19 | - | 4603.3 |
"-" indicates a tumor volume of more than 5000mm3。
The results of the drug effect are shown in the table. According to results, the curative effect of the docetaxel nano micelle on inhibiting the tumor growth is obviously better than that of docetaxel injection.
Example 13: cabazitaxel nano micelle prepared from different PEG-PE (polyethylene glycol-polyethylene) species
The prescription is shown in the table:
cabazitaxel nano micelle prescription prepared from different PEG-PE species
The preparation process comprises the following steps: weighing cabazitaxel and different types of PEG-PE according to the proportion of the formula, placing the cabazitaxel and different types of PEG-PE into a rotary evaporation bottle, adding a proper amount of ethanol, gently shaking to dissolve the drugs and the lipids to obtain a clear transparent solution, drying the organic solvent in the rotary evaporation bottle on a vacuum rotary evaporator at a rotating speed of 90r/min and a water bath temperature of 40 ℃, uniformly distributing the lipids and the drugs on the inner wall of the rotary evaporation bottle after the organic solvent is dried, then adding deionized water after the rotary evaporation bottle is cooled to room temperature, carrying out ultrasonic treatment in a water bath at 25 ℃ for 20min to completely dissolve a lipid membrane, then filtering by using a 220nm microporous filter membrane, then placing cabazitaxel nano micelle preparations with different drug-lipid ratios at room temperature (25 ℃), and observing the stability of the change investigation period of the preparation clarity, wherein the results are shown in the table.
Encapsulation efficiency and placement stability of cabazitaxel nano micelle preparation prepared from different PEG-PE (polyethylene glycol-polyethylene) types
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117045606A (en)* | 2022-05-07 | 2023-11-14 | 江苏百奥信康医药科技有限公司 | Bortezomib preparation and application thereof |
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