CN1289088C - Method for improving liposome enveloping rate of camptothecine or its derivatives - Google Patents
Method for improving liposome enveloping rate of camptothecine or its derivatives Download PDFInfo
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- CN1289088C CN1289088C CN 200410022041 CN200410022041A CN1289088C CN 1289088 C CN1289088 C CN 1289088C CN 200410022041 CN200410022041 CN 200410022041 CN 200410022041 A CN200410022041 A CN 200410022041A CN 1289088 C CN1289088 C CN 1289088C
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Abstract
The present invention discloses a method for enhancing the liposome enveloping rate of camptothecin or a derivative thereof. The method comprises the following steps: a mixed dry film or powder of camptothecin or a derivative thereof, phospholipid and cholesterol film material is prepared; liposome suspension of camptothecin or a derivative thereof is prepared; the pH value of the outer phase of the liposome suspension is regulated, and liposome of the camptothecin or the derivative thereof, which has high enveloping rate, is prepared. The medicine enveloping rate of the liposome of the camptothecin or the derivative thereof, which is prepared according to the method of the present invention, is from 75% to 100%, the biological availability is greatly improved, and the structure of anticancer activity ingredients is stable.
Description
Technical field
The present invention relates to the liposome of camptothecine or derivatives thereof, particularly improve the method for the liposome encapsulation of camptothecine or derivatives thereof.
Background technology
The camptothecine or derivatives thereof is the DNA synthetic inhibitor, is used for the treatment of multiple Cancerous disease.The camptothecine or derivatives thereof is site of action with the topoisomerase, target suppresses the synthetic of DNA and the medicine of performance antitumaous effect, be applicable to the tumor of the different speeds of growth, cytotoxicity is strong, and is obvious for the therapeutic effect of multiple malignant tumor such as gastric cancer, hepatocarcinoma, osteocarcinoma, leukemia, bladder cancer.The kind of camptothecine has camptothecine, hydroxy camptothecin, 7-ethyl-10-hydroxycamptothecine, 9-aminocamptothecin etc. in the market, by vein, oral, external, pulmonary's mucosa inhale as etc. the mode administration.
The camptothecine or derivatives thereof preparation of prior art for preparing, in clinical use, there is short, shortcoming such as bioavailability is low and toxic and side effects is obvious of half-life, and because the slightly solubility of camptothecine or derivatives thereof, multiselect is with the basic solvent system of high pH value in preparation process, active ingredient is reduced greatly, the active structure instability, and also targeting is also poor, thereby cause active anticancer to be restricted, reduced therapeutic index and safety index.For solving above-mentioned technological deficiency, people have adopted with liposomal encapsulated camptothecine and derivant thereof, improve the dissolubility of medicine, strengthen drug targeting, improve bioavailability and reduce toxic and side effects.The preparation method of conventional liposome comprises with volatile one or more ORGANIC SOLVENT MIXTURES dissolving multilamellar or double-deck constituents; under the condition of not phase-splitting, evaporate organic solvent then; dried lipoprotein mixture is deposited on the wall of reactor in the thin layer mode usually; reuse aqueous media aquation is sealed active matter; this aquation and to be encapsulated in dried fat layer very effective when very thin; this method has improved the stability and the targeting of active component effectively; but can only in laboratory, realize, be unsuitable for large-scale production.ZL95190971.1 discloses preparation method, device and the application that a kind of liposome precursor, efficient packet carry the power liposome.This method is dissolved in to form at least a organic solvent by one or more layers thin layer that forms lipoid substance has the liposome that efficient packet is carried power.This technology has solved the problem of large-scale production, but requires encapsulated medicine to have good hydrophilicity or lipotropy, to neither hydrophilic also alipotropic camptothecine or derivatives thereof is inapplicable.At above-mentioned defective, ZL95192734.5 discloses a kind of liposome that increases retention volume, this technology adopts neutral phospholipid that contains basic saturated fat acidic group and the charged phospholipid that contains basic satisfied fatty acid to prepare liposome membrane, solved water solublity non-electrolyte drug encapsulation effectively in liposome, improved the retention volume of active medicine, but the camptothecine or derivatives thereof has the chemical characteristic of slightly solubility, inapplicable this method.ZL95115156.8 discloses another kind of Injectable liposomal pharmaceutical preparation, said preparation is as the lipotropy of the liposome form of stabilizing agent, the liposome of slightly solubility reactive compound with short-chain fatty acid, improved the stability of reactive compound, but the envelop rate of liposome is still lower, only can reach 10%-30%.Therefore above-mentioned prior art all fails to solve the envelop rate problem of camptothecine or derivatives thereof liposome, and complicated process of preparation, the preparation instability that is obtained, and quality control difficulty height, the suitability for industrialized production difficulty is big.
Summary of the invention
Problem at above-mentioned prior art existence, the object of the invention is to provide a kind of envelop rate that improves the liposome of camptothecine and derivant thereof, use the camptothecine of the inventive method preparation and the liposome medicament active component Stability Analysis of Structures of derivant thereof, the medication targeting is strong, entrapment efficiency is higher than 75%, and is suitable for suitability for industrialized production.
The method of the liposome encapsulation of raising camptothecine or derivatives thereof provided by the invention is characterized in that may further comprise the steps:
Improve the method for the liposome encapsulation of camptothecine or derivatives thereof, it is characterized in that may further comprise the steps:
A) by following component (weight ratio) batching:
Camptothecine or derivatives thereof 0.01%～20%, phosphatidase 10 .1%～50%, cholesterol 0.05%～30%, water-soluble filler 0～50%, acid PH regulator and alkaline pH regulator are an amount of, and active component is the camptothecine or derivatives thereof;
(b) the mixture dry film or the powder of the liposome membrane material component of preparation camptothecine or derivatives thereof are dissolved in camptothecine or derivatives thereof, phospholipid, cholesterol in the organic solvent, obtain the mixture dry film or the powder of the liposome membrane material component of camptothecine or derivatives thereof with exsiccant method;
(c) the liposome suspension body of preparation camptothecine or derivatives thereof
Adding in the mixture dry film of the camptothecine or derivatives thereof that is obtained or powder and containing or do not contain water solublity filler, pH value in right amount is 7.5～13.0 alkaline PH regulator, after treating the mixture dry film or the abundant aquation of powder of camptothecine or derivatives thereof and liposome membrane material component, handling the acquisition particle diameter by particle diameter again is the liposome suspension body of 10～3000 nanometers;
(d) preparation has the liposome of the camptothecine or derivatives thereof of high envelop rate
Concentrate with the liposome suspension of drying means, add pH value to 2.5～6.5 that acid PH regulator is regulated the liposome suspension foreign minister then, make the liposome of camptothecine or derivatives thereof with high envelop rate with the camptothecine or derivatives thereof that obtained.
Further, camptothecin derivative described in the step (a) is selected from one or more in 10-hydroxycamptothecine, 7-ethyl-10-hydroxycamptothecine, 9-aminocamptothecin and the 9-nitrocamptothecin; Phospholipid is selected from one or more in phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, Ovum Gallus domesticus Flavus lecithin, hydrogenated soya phosphatide and the non-hydrogenated soya phosphatide described in the step (a); Water-soluble filler is selected from one or more in starch, dextran, lactose, maltose, sucrose, mannitol, gelatin, sorbitol, sodium chloride, polyvidon and the polyvinyl alcohol described in the step (a); Alkaline pH regulator described in the step (a) is selected from one or more of organic base, inorganic base, salt apoplexy due to endogenous wind, and acidic ph modifier is selected from one or more in organic acid, the mineral acid; Described organic base is selected from one or more in triethylammonium tetrakis, choline glycerophosphatide and the butylamine; Described inorganic base is selected from one or more in sodium hydroxide, potassium hydroxide, calcium hydroxide and the magnesium hydroxide; Described organic acid is selected from one or more in citric acid, lactic acid, arginine, glycine, serine, citric acid, formic acid and the acetic acid; Described mineral acid is selected from one or more in hydrochloric acid, phosphoric acid and the boric acid; Described salt is selected from one or more in sodium acetate, ammonium acetate, potassium acetate, sodium citrate, potassium citrate and the sodium lactate; Organic solvent is selected from one or more in straight chain alcohols, side chain alcohols, chloroform, dichloromethane and the normal hexane described in the step (b); Step (b) and (d) described in drying means can select in drying under reduced pressure, vacuum lyophilization, spray drying, centrifugal drying and the fluidized bed drying one or more for use; That particle diameter processing method described in the step (c) can be selected for use is ultrasonic, high-speed stirred, film extruding, high shear force homogenate and high pressure miniflow one or more.
The method of the liposome encapsulation of raising camptothecine or derivatives thereof provided by the invention, the method of the liposome suspension body of the adjusting camptothecine or derivatives thereof by adopting original creation and the pH value of the external phase of liposome suspension, the liposome active medicine envelop rate that makes prepared camptothecine or derivatives thereof by the 10%-30% of prior art to being higher than 75%, solved the low envelop rate technical barrier of liposome for preparing camptothecine or derivatives thereof in the prior art, and can realize industrial amplification production with slightly solubility characteristics.
The specific embodiment
Only the invention will be further described for the following examples, rather than restriction the present invention.
Embodiment 1
By prescription take by weighing camptothecine 62.5g, phosphatidylcholine 300g, cholesterol is 200g, with its mixing and to be dissolved in volume ratio be in 1: 12 liters of chloroform, the methanol mixed solution, fully stir and make its dissolving, adopt the vacuum decompression mode to remove organic solvent in the mixed solution then, obtain camptothecine mixture dry film; 437.5g starch added as water solublity fill agent and join 10 liters, pH value is in potassium hydroxide/boric acid solution of 12, make alkaline buffer solution, in the mixture dry film that is obtained, add this buffer solution and wash film, make its abundant aquation, form the big liposome of multilamellar, then according to the particle diameter requirement of vein and lung mucosa inhalation, adopting film extruding particle diameter processing mode to obtain particle diameter is the unilamellar liposome suspension liquid of 10-1000 nanometer, remove moisture in the buffer solution with the method for vacuum decompression then, make the volume that removes behind the moisture for removing 50% before the moisture, reuse 1N hydrochloric acid regulate to remove behind the moisture the liposome foreign minister to pH value be 5.5, the high liposome suspension body of sealing camptothecine.
Measure the liposome encapsulation of camptothecine
Get the liposome suspension 0.5ml that is obtained, with 10 times of liposome suspension dilutions, measure the medicine total amount, be designated as W with high-pressure liquid phase instrument (HPLC) with phosphate buffer
Always, ultrahigh speed is centrifugal then, gets centrifugation, measures camptothecine concentration with high-pressure liquid phase instrument (HPLC), thereby obtains the encapsulated amount of medicine, is designated as W
Bag, utilize formula envelop rate=(W
Always/ W
Bag), high-pressure liquid phase instrument (HPLC) is 95.5% according to the liposome encapsulation that the data computation of gathering to wrap the camptothecine of quilt.
Embodiment 2
By prescription take by weighing 10-hydroxycamptothecine 200g, phosphatidylcholine 300g, cholesterol is 500g, with its mixing and to be dissolved in volume ratio be in 1: 12 liters of chloroform, the methanol mixed solution, fully stir and make its dissolving, employing vacuum decompression method is removed the organic solvent in the mixed solution, obtains camptothecine mixture dry film; With 8 liters of pH value is that potassium hydroxide/boric acid alkaline buffer of 10 is as the pH regulator agent, in the mixture dry film that is obtained, add this pH regulator agent and wash film, make its abundant aquation, form the big liposome of multilamellar, then according to the particle diameter requirement of skin and oral administration, adopting film extruding particle diameter processing mode to obtain particle diameter is the unilamellar liposome suspension liquid of 1000-3000 nanometer, removes moisture in the buffer with the method for vacuum decompression, make cumulative volume be remove moisture preceding 50%.With 1N hydrochloric acid regulate to remove behind the moisture the liposome foreign minister to pH value be 4.5, the high liposome suspension of sealing camptothecine.
Measure the liposome encapsulation of hydroxy camptothecin
Get the liposome suspension 0.5ml of acquisition, with 10 times of liposome suspension dilutions, measure the medicine total amount, be designated as W with high-pressure liquid phase instrument (HPLC) with phosphate buffer
Always, ultrahigh speed is centrifugal then, gets centrifugation, measures camptothecine concentration, thereby obtains the encapsulated amount of medicine, is designated as W
Bag, utilize formula envelop rate=(W
Bag/ W
Always), high-pressure liquid phase instrument (HPLC) is 90.5% according to the liposome encapsulation that the data computation of gathering to wrap the camptothecine of quilt.
Embodiment 3
Take by weighing 7-ethyl-10-hydroxycamptothecine 100g, phosphatidylcholine 400g, cholesterol 200g by prescription, with its mixing and to be dissolved in volume ratio be in 1: 12 liters of the mixed solutions of chloroform, methanol, fully stir and make its dissolving, adopt the vacuum decompression method to remove organic solvent in the mixed solution then, obtain camptothecine mixture dry film; 300g mannitol added as water solublity fill agent to join 8 liters of pH value be in 13 potassium hydroxide/boric acid alkalescence pH regulator agent, make alkaline buffer solution, in the mixture dry film that is obtained, add this buffer solution and wash film, make its abundant aquation, form the big liposome of multilamellar, then according to the particle diameter requirement of skin and oral administration, adopting film extruding particle diameter processing mode to obtain particle diameter is the unilamellar liposome suspension liquid of 1000-3000 nanometer, remove moisture in the buffer with the method for vacuum decompression then, make the cumulative volume that removes behind the moisture for removing 20% before the moisture, with 1N hydrochloric acid regulate to remove behind the moisture the liposome foreign minister to pH value be 2.5, the high liposome suspension of sealing camptothecine.
Measure the liposome encapsulation of 7-ethyl-10-hydroxycamptothecine
Get the liposome suspension 0.5ml of acquisition, with 10 times of liposome suspension dilutions, measure the medicine total amount, be designated as W with high-pressure liquid phase instrument (HPLC) with phosphate buffer
Always, ultrahigh speed is centrifugal then, gets centrifugation, measures camptothecine concentration, thereby obtains the encapsulated amount of medicine, is designated as W
Bag, utilize formula envelop rate=(W
Bag/ W
Always), high-pressure liquid phase instrument (HPLC) is 78% according to the liposome encapsulation that the data computation of gathering to wrap the 7-ethyl-10-hydroxycamptothecine of quilt.
Claims (12)
1, improve the method for the liposome encapsulation of camptothecine or derivatives thereof, it is characterized in that may further comprise the steps:
(a) by following component (weight ratio) batching:
Camptothecine or derivatives thereof 0.01%～20%, phosphatidase 10 .1%～50%, cholesterol 0.05%～30%, water-soluble filler 0～50%, acid PH regulator, alkaline pH regulator are an amount of, and active component is the camptothecine or derivatives thereof;
(b) the mixture dry film or the powder of the liposome membrane material component of preparation camptothecine or derivatives thereof are dissolved in camptothecine or derivatives thereof, phospholipid, cholesterol in the organic solvent, obtain the mixture dry film or the powder of the liposome membrane material component of camptothecine or derivatives thereof with exsiccant method;
(c) the liposome suspension body of preparation camptothecine or derivatives thereof
Adding in the mixture dry film of the camptothecine or derivatives thereof that is obtained or powder and containing or do not contain water solublity filler, pH value in right amount is 7.5～13.0 alkaline PH regulator, after treating the mixture dry film or the abundant aquation of powder of camptothecine or derivatives thereof and liposome membrane material component, handling the acquisition particle diameter by particle diameter again is the liposome suspension body of 10～3000 nanometers;
(d) liposome of preparation with camptothecine or derivatives thereof of high envelop rate concentrates with the liposome suspension of drying means with the camptothecine or derivatives thereof that obtained, add acid PH regulator then and regulate liposome suspension foreign minister's pH value to 2.5～6.5, make the liposome of camptothecine or derivatives thereof with high envelop rate.
Camptothecin derivative is selected from one or more in 10-hydroxycamptothecine, 7-ethyl-10-hydroxycamptothecine, 9-aminocamptothecin and the 9-nitrocamptothecin described in the step (a).
2, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1 is characterized in that: phospholipid is selected from one or more in phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, Ovum Gallus domesticus Flavus lecithin, hydrogenated soya phosphatide and the non-hydrogenated soya phosphatide described in the step (a).
3, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1 is characterized in that: water-soluble filler is selected from one or more in starch, dextran, lactose, maltose, sucrose, mannitol, gelatin, sorbitol, sodium chloride, polyvidon and the polyvinyl alcohol described in the step (a).
4, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1, it is characterized in that: alkaline pH regulator described in the step (a) is selected from one or more of organic base, inorganic base, salt apoplexy due to endogenous wind, and acidic ph modifier is selected from one or more in organic acid, the mineral acid.
5, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 5, it is characterized in that: described organic base is selected from one or more in triethylammonium tetrakis, choline glycerophosphatide and the butylamine.
6, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 5, it is characterized in that: described inorganic base is selected from one or more in sodium hydroxide, potassium hydroxide, calcium hydroxide and the magnesium hydroxide.
7, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 5, it is characterized in that: described organic acid is selected from one or more in citric acid, lactic acid, arginine, glycine, serine, citric acid, formic acid and the acetic acid.
8, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 5, it is characterized in that: described mineral acid is selected from one or more in hydrochloric acid, phosphoric acid and the boric acid.
9, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 5, it is characterized in that: described salt is selected from one or more in sodium acetate, ammonium acetate, potassium acetate, sodium citrate, potassium citrate and the sodium lactate.
10, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1 is characterized in that: organic solvent is selected from one or more in straight chain alcohols, side chain alcohols, chloroform, dichloromethane and the normal hexane described in the step (b).
11, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1 is characterized in that: step (b) and (d) described in drying means can select in drying under reduced pressure, vacuum lyophilization, spray drying, centrifugal drying and the fluidized bed drying one or more for use.
12, the method for the liposome encapsulation of raising camptothecine or derivatives thereof according to claim 1 is characterized in that: the particle diameter processing method described in the step (c) can be selected one or more of ultrasonic, high-speed stirred, film extruding, high shear force homogenate and high pressure miniflow for use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022041 CN1289088C (en) | 2004-03-15 | 2004-03-15 | Method for improving liposome enveloping rate of camptothecine or its derivatives |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410022041 CN1289088C (en) | 2004-03-15 | 2004-03-15 | Method for improving liposome enveloping rate of camptothecine or its derivatives |
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| CN1562021A CN1562021A (en) | 2005-01-12 |
| CN1289088C true CN1289088C (en) | 2006-12-13 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101017164B (en) * | 2006-07-21 | 2011-08-31 | 上海中医药大学 | Determination method for entrapment efficiency of liposome |
| CN102670509B (en) * | 2011-03-08 | 2016-10-26 | 中国人民解放军军事医学科学院毒物药物研究所 | Liposomal formulation containing slightly solubility camptothecine and preparation method thereof |
| CN104031080B (en) * | 2014-06-20 | 2016-07-13 | 东北林业大学 | Silica nodule being loaded with 10-hydroxycamptothecine and preparation method thereof |
| US9675609B2 (en) * | 2015-11-11 | 2017-06-13 | Cao Pharmaceuticals Inc. | Nano- and micro-sized particles of 20-camptothecin or derivative thereof and pharmaceutical compositions containing same, and treatment of cancers therewith |
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Granted publication date: 20061213 Termination date: 20130315 |