CN103622911B - A kind of hard-soluble medicine liposome preparation method - Google Patents
A kind of hard-soluble medicine liposome preparation method Download PDFInfo
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- CN103622911B CN103622911B CN201310585359.5A CN201310585359A CN103622911B CN 103622911 B CN103622911 B CN 103622911B CN 201310585359 A CN201310585359 A CN 201310585359A CN 103622911 B CN103622911 B CN 103622911B
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Abstract
This method belongs to pharmaceutical formulating art, particularly a kind of preparation method of hard-soluble medicine liposome.The present invention adopts the separately blank liposome of preparation not with medicine, with drug suspension or drug solution, under uniform temperature, certain speed conditions, mix blank liposome and medicine subsequently, obtain hard-soluble medicine liposome through the method, its envelop rate >=90%, mean diameter≤150nm.The present invention adopts common process equipment, constant product quality, and method technique is simple, cost is low, solves hard-soluble medicine liposome and is difficult to industrialized technical barrier.
Description
Technical field
This method belongs to pharmaceutical preparation technology field, particularly a kind of preparation method of indissoluble medicaments liposome preparations.
Background technology
Liposome (liposome) is a kind of synthetic membrane.In water, phospholipid molecule hydrophilic head inserts in water, and liposome hydrophobic tail stretches to air, forms the spherical liposomes of double-deck fat molecule after stirring, and diameter 25 ~ 1000nm is not etc.Liposome can be used for transgenic, or the medicine of preparation, utilize liposome can with the feature of cell membrane fusion, medicine is sent into cell interior definition biology: when amphiphatic molecule is scattered in aqueous phase as phospholipid and sphingolipid, the hydrophobic tail of molecule is tended to flock together, and avoids aqueous phase, and hydrophilic head is exposed to aqueous phase, formed have bilayer structure vesicle, be called liposome.
Liposome is a kind of microgranule containing phospholipid fraction, and can be used as the carrier of insoluble drug, is a kind of new medicinal preparation.Pastille liposome is after intravenously administrable, mainly by reticuloendothelial system phagocytic, drug main to be put aside in the histoorgans such as liver, spleen, lung and bone marrow, change the distribution in vivo of encapsulated medicine, thus improve the therapeutic index of medicine, the therapeutic dose of medicine can be reduced and reduce the toxicity of medicine.
Liposome can be divided into by structure and particle diameter: unilamelar liposome, multilamelar liposome; Liposome can be divided into by performance: general liposome (comprising above-mentioned unilamelar liposome, multilamelar liposome and multiphasic liposomes etc.), property liposome, thermal sensitive liposome, pH sensitive liposome, ultrasound wave sensitive liposome, photosensitive liposomes and magnetic liposome etc.; Liposome can be divided into by charge: neutral liposome, elecrtonegativity liposome, electropositive liposome.
To present patent application, domestic and international research (patent CN101385715B, CN101507708B, CN1092044C, CN1236771C, CN100356919C) insoluble drug makes the substantially so-called Passive loading mode of method of liposome, namely be all that insoluble drug and phospholipid are mixed, then film forming, extruding finally makes liposome.In order to make the particle diameter of final products meet quality standard, needing repeatedly to extrude, easily causing the degraded of medicine, contaminant overstandard in the process.In general the product packaging rate that this technique obtains is not too high; And film-forming process is difficult to control, industrialization is made to become difficulty.
Summary of the invention
Technical problem to be solved by this invention is: in prior art, and adopt Passive loading mode, when preparing hard-soluble medicine liposome, envelop rate is low, complex process and be difficult to control.
For solving this technical problem, the technical solution used in the present invention is:
The present invention adopts the mode of Active loading to encapsulate insoluble drug first, the present invention is previously prepared not containing the blank liposome of active matter, and then prepare suspension or the solution of insoluble drug, both mixed under uniform temperature and stir speed (S.S.), mixed system is transformed into transparent or semitransparent shape by muddy shape.
Insoluble drug mentioned here is, in 100g water, dissolubility is lower than the medicine of 0.1g, comprise adefovir ester, docetaxel, camptothecine, methyldopa, dicoumarol, An Lu meter Te, paclitaxel, clofibrate, nifedipine, tolbutamide, insoral, rifamycin, the fast quinoline of sulfur azoles, etoposide, griseofulvin, famotidine, cyclosporin A, methotrexate, itraconazole, persantin, Ismipur, amiodarone, amphotericin B etc.
Said method comprises the following steps:
A, prepare blank liposome, and the mean diameter of blank liposome is controlled at nano-scale;
B, prepare insoluble drug solution, or prepare the suspension of insoluble drug;
C, the blank liposome will obtained in steps A, mix with the insoluble drug suspension obtained in step B or solution, obtained pastille liposome.
In steps A, the preparation method of blank liposome, comprises film ultrasound, injection method, reverse evaporation, fusion method, freeze-drying, ultrasonic dispersion, heterogeneous preparation method etc.,
Specifically: in steps A, phospholipid and additives, blank liposome is prepared under hot melt conditioned disjunction adds organic solvent condition, through buffer aquation, by mechanical means, blank liposome mean diameter is controlled at nano-scale (preferred mean diameter is at below 150nm), wherein phospholipid accounts for 0.1 ~ 50% of system by weight percentage
Wherein, additives comprise, cholesterol, vitamin E, 18-amine., di(2-ethylhexyl)phosphate spermaceti fat, soybean oil, Herba Amaranthi tricoloris oil generation or Fructus Canarii albi wet goods; Phospholipid comprises natural phospholipid, semi-synthetic phospholipid, synthetic phospholipid or its mixture, such as phosphatidylcholine, phosphatidylinositols, Phosphatidylserine, phosphatidyl glycerol, phosphatidic acid, two Laurel phosphatidylcholines, dimyristoyl phosphatidyl choline, DPPA, DOPC, DOPE, lecithin, soybean phospholipid, natural or synthesis cephalin, cuorin etc.
In step B, be scattered in by insoluble drug in organic solvent or buffer, controlling drug particles by mechanical means is fine powder state (preferred mean diameter is below 5 μm).
In foregoing, in steps A or step B, in the preparation of blank liposome, insoluble drug suspension or solution, described mechanical means, comprises ball-milling method, comminution by gas stream, high speed shear method, high pressure homogenization method, high pressure extrusion methods;
In steps A or step B, buffer can protect liposome with the preparation of monosaccharide and disaccharide, polysaccharide or its mixture, can also add simultaneously other buffer ions to, antioxidant, surfactant to strengthen the stability of insoluble drug dispersibility and liposome, as containing one or more buffer solution in histidine, valine, threonine, mannitol, sucrose, lactose, glucose, trehalose, arabic gum, xylitol, sorbitol, fructose; Surfactant is polyoxyethylene non-ionic surface active agent, the husky class nonionic surfactant in pool Lip river, scented hair oil used by women in former times class Determination of Polyoxyethylene Non-ionic Surfactants or its mixture,
In steps A or step B, machine solvent is selected from, one or more mixture in ethanol, methanol, ether, acetone, isopropyl alcohol, the tert-butyl alcohol, chloroform, dichloromethane, dimethyl sulfoxine, Methanamide.
In step C of the present invention, range of reaction temperature is 0 ~ 100 DEG C, and speed of agitator is may there is part organic solvent in 0 ~ 50000rpm, step C gained drug-loaded liposome, and the present invention adopts and intersects slipstream technological displacement and to publish originally medicine liposome solutions.In finished product, the residual quantity of organic solvent controls at 5%(weight fraction) below.
The finished product Liposomal formulation that the present invention finally obtains, degerming through 0.22 μm of membrane filtration, packaging or freezen protective.
As shown in Figure 1, liposomal particle size, according to upper figure matters, after preferably adopting ethanol injection to prepare blank liposome, is reduced to below 150nm by high pressure microjet, is convenient to industrialization degerming general preparation technology's flow chart by the present invention, for subsequent use.By insoluble drug by organic solvent dissolution or in being scattered in insoluble drug containing surfactant buffer, form mean diameter lower than the suspension of 5 μm.Above-mentioned two kinds of solution are mixed in proportion, stir, until system become translucent by muddiness or transparent liquid time, stop stirring, aseptic filtration, fill preservation or lyophilizing are preserved.
The preferred injection method of the present invention prepares blank liposome because there is underlying cause: can industrialization, and ethanol organic solvent is to environment, lower to human toxicity; After alcohol injection obtains liposome, process removed by ethanol.
Beneficial effect of the present invention is:
After insoluble drug makes suspension or solution, physical property has larger change, once add after in blank liposome, insoluble drug granule, more easily enter liposome, the mobility of liposomal phospholipids film, makes indissoluble granule can better enter liposome rete or inside, thus reaches the envelop rate higher than 90% simultaneously.Forming insoluble drug also uses " Active loading " mode entirely to seal.
The present invention adopts the mode of Active loading to encapsulate insoluble drug first, after liposome and medicament mixed, and can by the Liposomal formulation of aseptic filtration without the need to again being formed by high shear technology.Key issue in liposome production process will be solved: repeatedly extruding causes the problem such as drug degradation, contaminant overstandard; Liposomal formulation large-scale industrial production can be realized, avoid using more organic solvent simultaneously.
Accompanying drawing explanation
Fig. 1 is process chart of the present invention.
Fig. 2 is in embodiment 1, the grain-size graph of blank liposome.
Fig. 3 is in embodiment 1, is enclosed with the grain-size graph of the liposome of paclitaxel.
Fig. 4 is in embodiment 1, after lyophilizing is melted again, and Paclitaxel liposome grain-size graph.
Fig. 5 is in comparative example 1, after lyophilizing is melted again, and Paclitaxel liposome grain-size graph.
Fig. 6 is in comparative example 2, after lyophilizing is melted again, and AM Bison grain-size graph.
Detailed description of the invention
Involved by following examples, lipidosome Chinese traditional medicine thing content adopts HPLC method to measure; Particle diameter adopts laser device to measure; Envelop rate method of testing is as follows:
Post activates: get gel column G50 and rap and allow at the bottom of filler to pipe, remove red cap and put into 2ml centrifuge tube, add 500 μ l water, with 8000 revs/min of centrifugal 5min after 30min, after abandoning filtrate, add 500 μ l water again, with 8000 revs/min of centrifugal 5min after 15min, add 0.9% sodium chloride solution 500 μ l after abandoning filtrate with 8000 revs/min of centrifugal 5min, abandon filtrate, centrifugal 5min again, changes centrifuge tube for subsequent use.
Sample thief is appropriate, and adding water, to make in every 1ml solution containing paclitaxel be the suspension of 2mg.
Precision measures suspension 200 μ l on the gel column activated, after 10 minutes, centrifugal 5 minutes with 8000 revs/min, add 500 μ l water more centrifugal 5 minutes with 8000 revs/min on gel column, merging filtrate is in 10ml volumetric flask, with acetic acid: methanol (1:200) washing centrifuge tube 3 times is also transferred in volumetric flask, add with acetic acid: after methanol (1:200) dissolves, transfer is dissolved and is settled to scale, shake up, HPLC method is adopted to measure, precision measures 10 μ l injection liquid chromatographies, and record chromatogram, peak area is designated as A
encapsulating.
Above-mentioned gel column is more successively with 25% ethanol 2ml, 50% ethanol 2ml, the centrifugal eluting of dehydrated alcohol 4ml gradation, eluent is all transferred in another 10ml volumetric flask, dehydrated alcohol is diluted to scale, shake up, HPLC method is adopted to measure, precision measures 10 μ l injection liquid chromatographies, and record chromatogram, peak area is designated as A
free.
Precision measures in suspension 200 μ l to 10ml volumetric flask, adds acetic acid: methanol (1:200) is diluted to scale, shakes up, and according to the chromatographic process under assay item, precision measures 10 μ l injection liquid chromatographies, and record chromatogram, peak area is designated as A
always medicine.
According to formulae discovery envelop rate and the post response rate:
Reduced mechanical model:
A
encapsulating: for post is separated encapsulating sample through acetic acid: sample solution peak area after methanol (1:200) dilution
A
free: be post separated free sample sample solution peak area after dehydrated alcohol dilution
A
total medicine: for non-suspended liquid is through with acetic acid: sample solution peak area after methanol (1:200) dilution
Embodiment 1
Preparation process A
Blank liposome prescription:
Phospholipid, soybean oil chloroform are dissolved, be placed on Rotary Evaporators, removing chloroform, forms immobilized artificial membrane.Mannitol is soluble in water, be added in immobilized artificial membrane, by immobilized artificial membrane eluting, through high pressure homogenize instrument, mean diameter controlled at below 150nm for subsequent use.
The grain-size graph of blank liposome as shown in Figure 2.
Preparation process B
Paclitaxel solution prescription:
Paclitaxel 0.4g
Ethanol 2g
Paclitaxel is dissolved in ethanol.
Preparation process C
Add the obtained blank liposomes liquid solution of steps A in stepb (in step B, paclitaxel can be dissolved in ethanol, when alcoholic solution is mixed with water, paclitaxel is water insoluble and separate out, its granule is very little, reaches micron-scale), at room temperature, stir under 200rpm, system is by muddy bleach shape liquid.Adopt slipstream technology of intersecting, removing ethanol makes its residual quantity below 5%.Degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing.
Accelerated stability data
Embodiment 2
Preparation process A
Blank liposome prescription:
Phospholipid, Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, cholesterol are added ethanol 60 DEG C of heating for dissolving, by trehalose, histidine is soluble in water, ph is adjusted to be 6.5 with hydrochloric acid, be heated to 65 DEG C, above-mentioned alcoholic solution is injected, after stirring 30min, through high pressure homogenize instrument, mean diameter is controlled at below 180nm for subsequent use.
Preparation process B
Taxotere solution prescription:
Taxotere 0.6g
Ethanol 2g
Taxotere is dissolved in ethanol.
Preparation process C
Add blank liposomes liquid solution (preparation principle of insoluble drug suspension or solution as shown in Example 1) obtained in steps A in stepb, at 4 DEG C, stir under 100rpm, system is by muddy bleach shape liquid, after intersection tangential flow device carries out buffer exchange (residual quantity of ethanol controls below 5%), degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing.
Accelerated stability data
Embodiment 3
Preparation process A
Blank liposome prescription:
By phospholipid, phosphatidyl glycerol, cholesterol adds methanol, dichloromethane dissolves, and obtains organic solution; By sucrose, sodium succinate is soluble in water, adjusts ph to be 5.5, be heated to 35 DEG C with hydrochloric acid.Above-mentioned organic solution slowly injected, after stirring 30min, vacuum reclaims organic solvent, then through high pressure homogenize instrument, controls at below 100nm for subsequent use by mean diameter.
Preparation process B
Amphotericin B suspension prescription:
Amphotericin B 1g
Sodium deoxycholate 0.1g
Water 10g
Sodium deoxycholate is dissolved in the water, adds amphotericin B, through colloid mill grinding, make amphotericin B mean particle size below 5 μm.
Preparation process C
Add blank liposomes liquid solution obtained in steps A in stepb, at 70 DEG C, stir under 20000rpm, system is by the translucent liquid of the muddy yellowing of yellow, degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing.Obtain AM Bison, mean diameter 120nm, envelop rate > 90%.
Accelerated stability data
Comparative example 1:(does not control the particle diameter of blank liposome)
Preparation process A
Blank liposome prescription:
Phospholipid, soybean oil chloroform are dissolved, be placed on Rotary Evaporators, removing chloroform, forms immobilized artificial membrane.Mannitol is soluble in water, be added in immobilized artificial membrane, obtain blank liposomes liquid solution.
Preparation process B
Paclitaxel solution prescription:
Paclitaxel 0.4g
Ethanol 2g
Paclitaxel is dissolved in ethanol.
Preparation process C
Add in the obtained blank liposomes liquid solution of steps A in stepb, mix homogeneously, stirs 60 minutes under 200rpm.Adopt slipstream technology of intersecting, removing ethanol makes its residual quantity below 5%.Degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing.
The liposome medicament that above-mentioned preparation method obtains, envelop rate is 62.4%, and mean diameter is 457nm.
In this comparative example, after lyophilizing is melted again, Paclitaxel liposome grain-size graph as shown in Figure 5.
Comparative example 2:(does not control the particle diameter of blank liposome, and does not control the particle diameter of insoluble drug)
Preparation process A
Blank liposome prescription:
By phospholipid, phosphatidyl glycerol, cholesterol adds methanol, dichloromethane dissolves, and obtains organic solution; By sucrose, sodium succinate is soluble in water, adjusts ph to be 5.5, be heated to 35 DEG C with hydrochloric acid.Above-mentioned organic solution slowly injected, after stirring 30min, vacuum reclaims organic solvent, obtains blank liposomes liquid solution.
Preparation process B
Amphotericin B suspension prescription:
Amphotericin B 1g
Sodium deoxycholate 0.1g
Water 10g
Sodium deoxycholate is dissolved in the water, adds amphotericin B, stir.
Preparation process C
Add blank liposomes liquid solution obtained in steps A in stepb, at 70 DEG C, stir 60 minutes under 20000rpm, degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing.
The liposome medicament that above-mentioned preparation method obtains, envelop rate is 58.6%, and mean diameter is 421nm.
In this comparative example, after lyophilizing is melted again, AM Bison grain-size graph as shown in Figure 6.
Claims (1)
1. a hard-soluble medicine liposome preparation method, is characterized in that: described method is,
Steps A
Blank liposome prescription is
Phospholipid, Polyethylene Glycol PHOSPHATIDYL ETHANOLAMINE, cholesterol are added ethanol 60 DEG C of heating for dissolving; By trehalose, histidine is soluble in water, adjusts ph to be 6.5, be heated to 65 DEG C, injected by above-mentioned alcoholic solution with hydrochloric acid, after stirring 30min, through high pressure homogenize instrument, mean diameter is controlled at below 180nm, obtains blank liposomes liquid solution;
Step B
Taxotere solution prescription is
Taxotere 0.6g
Ethanol 2g
Taxotere is dissolved in ethanol;
Step C
Add blank liposomes liquid solution obtained in steps A in stepb, at 4 DEG C, stir under 100rpm, system is by muddy bleach shape liquid, after intersection tangential flow device carries out buffer exchange, the residual quantity of ethanol controls below 5%, degerming through 0.22 μm of membrane filtration, subpackage, lyophilizing, obtained pastille liposome.
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| CN106619516B (en) * | 2016-10-10 | 2020-07-03 | 常州金远药业制造有限公司 | Preparation method of small molecule targeted drug liposome preparation |
| LT3532067T (en) | 2016-10-28 | 2022-08-25 | Les Laboratoires Servier | Liposomal formulation for use in the treatment of cancer |
| EP3634385A4 (en) * | 2017-05-12 | 2021-03-03 | Curinanorx, LLC | Methods for the preparation of liposomes comprising drugs |
| CN108721644B (en) * | 2018-06-05 | 2021-06-08 | 常州金远药业制造有限公司 | Preparation method of taxane medicine liposome |
| CN116036032B (en) * | 2023-03-30 | 2023-06-13 | 山东新时代药业有限公司 | Famotidine tablet, preparation method and application |
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| CN101816640A (en) * | 2010-04-16 | 2010-09-01 | 海南美大制药有限公司 | Prasugrel liposome solid preparation |
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| CN101816640A (en) * | 2010-04-16 | 2010-09-01 | 海南美大制药有限公司 | Prasugrel liposome solid preparation |
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