CN102716095B - Paclitaxel vesicle type phospholipid gel injection - Google Patents
Paclitaxel vesicle type phospholipid gel injection Download PDFInfo
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Abstract
The invention discloses paclitaxel vesicle type phospholipid gel injection and a preparation method of the paclitaxel vesicle type phospholipid gel injection. The paclitaxel vesicle type phospholipid gel injection is prepared from paclitaxel, dimyristoyl phosphatidyl ethanolamine, stigmasterol and trehalose according to a specific weight ratio. The paclitaxel vesicle type phospholipid gel injection disclosed by the invention has good preparation stability, and the vesicle type phospholipid gel maintains a good encapsulation rate after long-period storage. The paclitaxel vesicle type phospholipid gel injection and the preparation method have the advantages that the quality of preparation products is good, the bioavailability of the medicine is improved, the toxic and side effects are reduced, and in addition, the preparation method is simple and is suitable for industrial mass production.
Description
Technical field
The present invention relates to the novel injection of a kind of paclitaxel and method for making thereof, be specifically related to a kind of paclitaxel vesicle type phospholipid gel injection and method for making thereof, belonged to technical field of medicine.
Background technology
Paclitaxel (Paclitaxel) product another name: taxol, purple plain, special plain, chemical name: 5 β, 20-epoxy-1,2 α, 4,7 β, 10 β, 13 α-hexahydroxy taxane-11-alkene-9-ketone-4,10-diacetate esters-2-benzoate-13[(2 ' R, 3 ' S)-N-benzoyl-3-phenylisoserine ester].White crystals body powder.Odorless, tasteless.Water insoluble, in methanol, ethanol, acetone or chloroform, dissolve slightly soluble in ether.Relative molecular mass: 853.92, molecular formula: C47H51NO14; Structural formula is as follows:
Americanized scholar's Giovanni (M.C.Wani) in 1963 and Wal (Monre E.Wall) are first from a kind of crude extract that has been separated to paclitaxel US West's fully stocked wood in peaceful red deal (Pacific Yew) bark of appellation and timber that is grown in.In screening experiment, Wani and Wal l find that the paclitaxel crude extract has very high activity to the Mus tumor cell of isolated culture, and begin to separate this active ingredient.Because this active ingredient content in plant is extremely low, up to 1971, they are chemistry professor Mu Kefaer (Andre T.McPhail) cooperation of same Du Ke (Duke) university, determined the chemical constitution of this active ingredient by the x-ray analysis---a kind of tetracyclic diterpene chemical compound, and its called after paclitaxel (taxol).
Paclitaxel is the secondary metabolite of a kind of complexity in the Chinese yew genus plants, also is that the only a kind of of present institute familiar with understanding can promote microtubule polymerization and the stable medicine of polymerization microtubule.Isotopic tracing shows that paclitaxel only is attached on the microtubule of polymerization, not with unpolymerized tubulin dimer reaction.Can accumulate a large amount of microtubules in cell behind the cells contacting paclitaxel, the various functions of cell have been disturbed in the accumulation of these microtubules, particularly make cell division stop at mitotic phase, have blocked the proper splitting of cell.By II-III clinical research, paclitaxel mainly is applicable to ovarian cancer and breast carcinoma, and pulmonary carcinoma, colorectal cancer, melanoma, incidence cancer, lymphoma, cerebroma are also had certain curative effect.
Pharmacokinetics: vein gives paclitaxel, and drug plasma concentration is diphasic curve.This product protein binding rate 89%～98%, paclitaxel is at liver metabolism.Paclitaxel enters intestinal mainly at liver metabolism with bile, excretes (〉 90% through feces), the good clinical using value is arranged.
Pharmacological toxicology: this product is novel anti microtubule medicine, suppresses depolymerization by promoting tubulin polymerization, keeps tubulin stable, suppresses cell mitogen.Experiment in vitro proof paclitaxel has significant radiosensitizing effect, may be that cell was terminated in radiotherapy sensitive G 2 and M phase.
The paclitaxel of listing mostly is injection at present, because paclitaxel is insoluble in water, is that solvent is made with polyoxy ethyl Oleum Ricini and ethanol all mostly, and polyoxy ethyl Oleum Ricini has antigenicity, can be directed at the receiver and severe anaphylactic reaction occur.The present invention is developed into a kind of paclitaxel injection that does not contain polyoxy ethyl Oleum Ricini, and goods adopt the specific process PROCESS FOR TREATMENT
Patent CN101190214A discloses a kind of paclitaxel injection and preparation method thereof, and (1) adds PEG400 150400ml after getting dehydrated alcohol 200-400ml, normal saline 200-400ml stirring, stirs; (2) in (1), add Tween 80 20-40g, stir evenly, add the 0.2-0.5g needle-use activated carbon, heat 35-40 ℃, coarse filtration, pH value 3-5 filters, and embedding is at the 100-125 ℃ of 20-35min that sterilizes down; (3) with after the infusion bottle cleaning, in 230-260 ℃ of dry 35min, top plug sterilization; (4) get paclitaxel 0.6-3g and add dissolving in (2), aseptic filtration under aseptic purification condition, pours into infusion bottle, seals namely.Though this method has been avoided polyoxy ethyl Oleum Ricini, a large amount of surfactant of its solvent, cosolvent and organic solvent still can bring injury to human body.
In the pharmaceutical carrier induction system, the research of submicrons such as microemulsion, microsphere, nanoparticle, liposome, vesicle type phospholipid gel, pharmacosomes has become field very active in the novel pharmaceutical formulation research.Drug encapsulation can be changed medicine distribution in vivo in these submicrons, increase medicine in the abundance of target organ, thereby improve curative effect, alleviate toxic and side effects.
Patent CN101057831A, CN101011357A, CN101322689A, CN101889982A, CN101584663A, CN101744767A and patent CN101883557A, various Paclitaxel liposome preparations are disclosed.For example patent CN101011357A discloses a kind of method of preparation of Paclitaxel liposome preparation, adopt film dispersion method or spray drying method for preparation long-circulating paclitaxel liposome, be stabilizing agent with cholesterol, distearyl acyl group phosphatldylcholine and tetradecylic acid, sucrose is freeze drying protectant, and chloroform methanol is organic solvent; And adopt amphipathic ethylene glycol derivative modified liposome film, adopt the method for extruding or high pressure homogenize to make liposome particle diameter≤100nm, envelop rate 〉=85% simultaneously.These methods have improved with respect to common paclitaxel injection preparation a lot, its preparation toxicity is little stable height, but also be not enough to meet clinical needs, vesicle type phospholipid gel injection stability with respect to Paclitaxel liposome preparation bigger raising of having got back of our invention also overcomes the toxicity that liposome brings because of phospholipid oxidation.
Conventional liposome has experienced many historical development since half a century.But the inferior position of dosage form itself also makes it face the test of Yan Jun, aspects such as drug leakage, system stability decreases all become the weak point that limits its development, vesicle type phospholipid gel (Vesicular phospholipid gel on the basis of conventional liposome, VPG) as a kind of novel medicament transmission system, show special advantages at aspects such as control drug release, raising anti-tumor activity, increase medicine stabilities.
Vesicle type phospholipid gel is a kind of semisolid phospholipid disperse system.Similar vesicle on the form, and be different from traditional lipidosome gel and conventional liposome cell structure.Because formed unique three-dimensional netted stereochemical structure, VPG can be used as " the storage storehouse " of medicine, thus the control drug release.The phospholipid denseness that increases is dwindled the interval between adjacent vesicle, and final result is in the vesicle and all contains the water of certain volume between vesicle, so when pressure sterilizing and long term store, stability is unaffected.
In recent years, continuous progress along with biotechnology, preparation technology is progressively perfect for vesicle type phospholipid gel, vesicle type phospholipid gel mechanism of action is further illustrated, vesicle type phospholipid gel is fit to vivo degradation, avirulence and non-immunogenicity in addition, particularly great number tested data proof vesicle type phospholipid gel can improve the Drug therapy index, reduces drug toxicity and reduce drug side effect as pharmaceutical carrier, and reduces advantage such as drug dose.
CN102125517A discloses a kind of low concentration vesicle type phospholipid gel preparation of small-molecule peptide medicine, and the preparation method of this small-molecule peptide medicine vesicle type phospholipid gel preparation and the influence of phospholipid concentration are provided.Vesicle type phospholipid gel is different from the disclosed liposome gel of CN101883557A, also be different from CN101229150A, CN101249067A, CN101283976A, CN101336891A, CN102335455A, KR100746962B gel injection, these patent documentations are with gel-type vehicle and active component or its liposome mix homogeneously, obtain gel, with vesicle type phospholipid gel of the present invention be diverse dosage form.
Zhang Ruizhi etc. are in vesicle type phospholipid gel progress, " Chinese journal of Practical Pharmacy " in July, 2008, the 6th volume the 4th interim concept of introducing vesicle type phospholipid gel, the principle that forms, preparation method and application progress etc., and illustrate vesicle type phospholipid gel as a kind of novel drug-supplying system in conjunction with instantiation, at the control drug release, improve anti-tumor activity, increase aspects such as medicine stability and show special advantages.All mention in the application or each list of references of quoting is introduced in full at this, as a reference.
Summary of the invention
To achieve these goals, big quantity research and realization that the inventor carries out, find the paclitaxel of specified weight proportioning, two myristoyl PHOSPHATIDYL ETHANOLAMINE, stigmasterol and trehalose can be made excellent paclitaxel vesicle type phospholipid gel injection, wherein, envelop rate height as the paclitaxel of active constituents of medicine, vesicle type phospholipid gel particle diameter is little and be evenly distributed, compare with paclitaxel injection of the prior art, the retention time significant prolongation of the active constituents of medicine of preparation of the present invention in the body circulation, the biocompatibility height of medicine, bioavailability obviously improves, and curative effect obviously improves.
The purpose of this invention is to provide a kind of paclitaxel vesicle type phospholipid gel injection, it is mainly made by the composition of following weight proportion:
Preferably: the weight ratio between stigmasterol and the two myristoyl PHOSPHATIDYL ETHANOLAMINE is 1:3-2:5, and the ratio between paclitaxel and the two myristoyl PHOSPHATIDYL ETHANOLAMINE is 1:50-1:60.
As the preferred embodiments of the invention, described paclitaxel vesicle type phospholipid gel injection, mainly made by the composition of following weight proportion:
Generally speaking, comprise PHOSPHATIDYL ETHANOLAMINE as the material that is used to form vesicle type phospholipid gel, phosphatidyl glycerol, Phosphatidylserine, phosphatidylinositols, phosphatidylcholine, Ovum Gallus domesticus Flavus lecithin, hydrogenated yolk lecithin, EPG, egg yolk lecithin acyl serine, egg yolk lecithin acyl inositol, soybean lecithin, hydrogenated soy phosphatidyl choline, hydrolecithin, lecithin acyl glycerol, lecithin acyl serine, lecithin acyl inositol, the dioleoyl phospholipid phatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidyl choline, two myristoyl phosphatidylcholines, two Laurel phosphatidyl cholines, DOPG, the distearyl phosphatidyl glycerol, two palmityl phosphatidyl glycerols, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two lauroyl phosphatidyl glycerols, DOPE (DOPE), DSPE (DSPE), two myristoyl PHOSPHATIDYL ETHANOLAMINE (DMPE), two palmityl PHOSPHATIDYL ETHANOLAMINE (DPPE), cholesterol, β-sitoesterol, stigmasterol, cholesterol succinate and polyglycol derivatization phospholipid be DSPE-Macrogol 2000 for example; Two soft ester acyl gallbladder phospholipid-Macrogol 2000s; Hydrogenated soya phosphatide phatidylcholine-Macrogol 2000; Dioleoyl phospholipid phatidylcholine-Macrogol 2000 etc.
The inventor is through research in earnest for a long time, through a large amount of screening experiment, finishing screen is chosen the combination of DSPE and these two kinds of materials of stigmasterol, find the combination of DSPE and two kinds of materials of stigmasterol unexpectedly, stability and the not good technical problem of envelop rate of vesicle type phospholipid gel have been solved, obtained beyond thought preparation effect, thereby superior in quality vesicle type phospholipid gel is provided.Though do not want to be bound by theory, effect of the present invention may be the common and/or synergistic result of DSPE, two kinds of materials of stigmasterol, but not any or multiple vesicle type phospholipid gel material of DSPE and stigmasterol combination does not all obtain superior in quality stable paclitaxel vesicle type phospholipid gel.
In paclitaxel vesicle type phospholipid gel injection of the present invention, for the paclitaxel of 1 weight portion, the consumption of two myristoyl PHOSPHATIDYL ETHANOLAMINE is the 40-70 weight portion.If the consumption of two myristoyl PHOSPHATIDYL ETHANOLAMINE is lower than 40 weight portions, then the stability of the vesicle type phospholipid gel of Xing Chenging can reduce significantly; Otherwise if the consumption of the consumption of two myristoyl PHOSPHATIDYL ETHANOLAMINE is higher than 70 weight portions, then the envelop rate as the paclitaxel of active constituents of medicine also can descend thereupon, and the quality of injection and curative effect reduce.Preferably for the paclitaxel of 1 weight portion, the consumption of two myristoyl PHOSPHATIDYL ETHANOLAMINE is the 50-60 weight portion.
In paclitaxel vesicle type phospholipid gel injection of the present invention, stigmasterol is used for regulating the stability of vesicle type phospholipid gel.
Stigmasterol (Stigmasterol) is to extract a plant sterols that forms from the concise product of soybean oil, and is without any side effects, is cholesterol reducing, prevents and treats cardiovascular disease, anticancer desirable material.As a kind of natural product, the stigmasterol source is abundant, low price.With cholesterol seemingly, stigmasterol also can be regulated the stability of phosphatidylinositols film, and its regulating action effect to stability is better than cholesterol.
Stigmasterol and cholesterol seemingly combine with two myristoyl PHOSPHATIDYL ETHANOLAMINE, stop it to be condensed into crystal structure.Mix two myristoyl PHOSPHATIDYL ETHANOLAMINE bilayers, be similar to " buffer agent " and equally play the effect of regulating membrane structure " flowability ".When being lower than phase transition temperature, can make film reduce ordered arrangement, increase mobile; When being higher than phase transition temperature, can increasing the ordered arrangement of film, thereby reduce the flowability of film.Liposome bimolecular tunic is solidified, thereby reduce the generation of free radical, reduce oxidation level, liposome stability is significantly strengthened.Its source is abundant, low price.
The inventor when stigmasterol and two myristoyl PHOSPHATIDYL ETHANOLAMINE weight ratios are preferably 1:3-2:5, can form stable paclitaxel vesicle type phospholipid gel through discovering.When with stigmasterol and two myristoyl PHOSPHATIDYL ETHANOLAMINE weight ratios during less than 1:3, membrane stability reduces, and paclitaxel is easy to seepage; When stigmasterol and two myristoyl PHOSPHATIDYL ETHANOLAMINE weight ratios during greater than 2:5, paclitaxel vesicle type phospholipid gel membrane fluidity is too high, and the paclitaxel that is wrapped in the vesicle type phospholipid gel is easy to discharge.In addition, discover that when stigmasterol and two myristoyl PHOSPHATIDYL ETHANOLAMINE weight ratios were 1:3-2:5, formed vesicle type phospholipid gel toxicity was minimum.
On the other hand, the inventor discovers, in paclitaxel vesicle type phospholipid gel injection of the present invention, for the paclitaxel of 1 weight portion, the consumption of two myristoyl PHOSPHATIDYL ETHANOLAMINE is the 50-60 weight portion, when stigmasterol is 20 weight portions, the envelop rate height of formed paclitaxel vesicle type phospholipid gel injection.
Discover that when the paclitaxel that uses above-mentioned specified quantitative, two myristoyl PHOSPHATIDYL ETHANOLAMINE, stigmasterol, can obtain colory paclitaxel vesicle type phospholipid gel, its toxicity is low, stability is high, the bioavailability height.
In paclitaxel vesicle type phospholipid gel injection of the present invention, use trehalose as excipient, be used to form stable injection.
Trehalose be by two glucose molecules with α, α, 1; the nonreducing sugar that the 1-glycosidic bond constitutes; self property is highly stable, and its most tangible character is that the biomembranous ability of protection is arranged under anhydrous condition, even make vesicle type phospholipid gel also keep complete form under the situation of dehydration.So in paclitaxel vesicle type phospholipid gel injection of the present invention, trehalose can effectively be protected form and the stability of vesicle type phospholipid gel granule, further improves the stability of vesicle type phospholipid gel injection.
Paclitaxel vesicle type phospholipid gel injection of the present invention, its specification is 5ml:30mg, 10ml:60mg, 25ml:150mg or 30mg/ bottle.
The present invention also provides a kind of method for making of paclitaxel vesicle type phospholipid gel injection, it is characterized in that comprising the steps:
(1) under nitrogen protection, paclitaxel, two myristoyl PHOSPHATIDYL ETHANOLAMINE and stigmasterol are dissolved in the proper amount of solvent, about 45 ℃ were descended slow magnetic agitation 15-30 minute, and obtained taxusol-lipid solution;
(2) with above-mentioned solution lyophilization, complete until drying, obtain forming loose spongiform drying solid;
(3) under nitrogen protection with above-mentioned solid dispersion in aqueous trehalose, ultrasonic 15～30 minutes of 400W-600W, 200bar does the gradient homogenizing 6～8 times preferred 7 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with the lyophilization of paclitaxel vesicle type phospholipid gel, the lyophilized powder direct packaging obtains paclitaxel vesicle type phospholipid gel freezing-dried powder injection; Perhaps with the lyophilization of paclitaxel vesicle type phospholipid gel, add the back packing that is uniformly dispersed of a certain amount of water for injection then, namely get paclitaxel vesicle type phospholipid gel injection.
In a preferred embodiment of paclitaxel vesicle type phospholipid gel injection preparation method of the present invention, solvent described in the step (1) is selected from one or more in water for injection, ethanol, chloroform, dichloromethane, methanol, n-butyl alcohol, isopropyl alcohol, isobutanol, benzyl alcohol, the tert-butyl alcohol, the chloroform, the mixed solvent of preferred water for injection, ethanol and the tert-butyl alcohol; More preferably described mixed solvent is that the volume ratio of water for injection, ethanol and the tert-butyl alcohol is the solution of 1:2:7.
In a preferred embodiment of paclitaxel vesicle type phospholipid gel injection preparation method of the present invention, cryodesiccated process is described in the step (2): under-60 ℃ of conditions freezing 5-8 hour, be warming up to 25 ℃ with 5-10 ℃/hour speed again, at last at 25 ℃ of dry 2-4 hours to fully dry.
In a preferred embodiment of paclitaxel vesicle type phospholipid gel injection preparation method of the present invention, cryodesiccated process was described in the step (4) :-60 ℃～-70 ℃ pre-freezes 2～3 hours, then freezing 6～8 hours at-40 ℃～-50 ℃, sublimed up into 20 ℃～25 ℃ through 18～24 hours again, extremely drying was complete in 1～3 hour 25 ℃～30 ℃ dryings at last.
Discover, existing multicell structure also has single cell structure in the vesicle type phospholipid gel, the kind of phospholipid and concentration and high pressure homogenize condition all influence the form of VPG, control above-mentioned factor, can obtain the sizeable three-dimensional netted vesicle phospholipid gel of the uniform vesicle of cell structure.VPG can be used as drug release " storage storehouse "; Under the situation that adds certain aqueous medium, VPG can be transformed into common small unilamellar vesicle.Diffusion by medicine when the release of vesicle type phospholipid gel Chinese medicine is main and the corrosion of VPG, so the kind of phospholipid and concentration and vesicle size have decisive role to the release of medicine.By the paclitaxel vesicle type phospholipid gel of the inventive method preparation, particle size distribution is even, and this is one of its factor that metabolic rate is low in vivo, bioavailability is high.This vesicle type phospholipid gel solid preparation is under the situation that adds certain aqueous medium, for example according under regulation aqueous medium and the experiment condition in 2010 editions appendix dissolutions of Chinese Pharmacopoeia or the drug release determination, stir or jolting, can be transformed into common small unilamellar vesicle after the VPG aquation.
The paclitaxel vesicle type phospholipid gel injection that makes by the inventive method, increased the retention time of medicine in the body circulation, improved bioavailability of medicament, reduced toxic and side effects, curative effect obviously improves, improved the long-time stability of preparation, and method for making is simple, is suitable for industrialized great production.
Description of drawings
Fig. 1 be the paclitaxel vesicle type phospholipid gel injection for preparing among embodiment 1-4 and the Comparative Examples 1-4 and commercially available paclitaxel injection concentration and time relation curve chart.
Wherein:
The specific embodiment
Further specify the present invention by the following examples, but should not be construed as limitation of the present invention.
The preparation of embodiment 1 paclitaxel vesicle type phospholipid gel injection
Prescription: (1000 bottles)
Preparation technology
(1) under nitrogen protection, the volume ratio that paclitaxel 30g, two myristoyl PHOSPHATIDYL ETHANOLAMINE 1500g and stigmasterol 600g is dissolved in water for injection, ethanol and the tert-butyl alcohol is in the 3000ml solution of 1:2:7, slow magnetic agitation is 25 minutes under about 45 ℃, obtains taxusol-lipid solution;
(2) with above-mentioned solution under-60 ℃ of conditions freezing 7 hours, be warming up to 25 ℃ with 10 ℃/hour speed again, 25 ℃ of dryings 4 hours, obtain forming loose spongiform drying solid at last;
(3) under nitrogen protection, above-mentioned solid dispersion is contained in the aqueous solution of trehalose 300g in 4000ml, ultrasonic 30 minutes of 400W, 200bar does the gradient homogenizing 8 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with paclitaxel vesicle type phospholipid gel through the deep bid lyophilization (-70 ℃ of pre-freezes 2 hours, then freezing 6 hours at-50 ℃, sublimed up into 20 ℃ through 18 hours again, at last 25 ℃ of dryings 3 hours), add water for injection again and be uniformly dispersed to 5L, the packing of 5ml/ bottle namely gets paclitaxel vesicle type phospholipid injection.
The preparation of embodiment 2 paclitaxel vesicle type phospholipid gel injections
Prescription: (1000 bottles)
Preparation technology
(1) under nitrogen protection, the volume ratio that paclitaxel 60g, two myristoyl PHOSPHATIDYL ETHANOLAMINE 3600g and stigmasterol 1200g is dissolved in water for injection, ethanol and the tert-butyl alcohol is in the 6000ml solution of 1:2:7, slow magnetic agitation is 20 minutes under 45 ℃, obtains taxusol-lipid solution;
(2) with above-mentioned solution under-60 ℃ of conditions freezing 6 hours, be warming up to 25 ℃ with 5 ℃/hour speed again, 25 ℃ of dryings 3 hours, obtain forming loose spongiform drying solid at last;
(3) under nitrogen protection, above-mentioned solid dispersion is contained in the aqueous solution of trehalose 600g in 9000ml, ultrasonic 20 minutes of 600W, 200bar does the gradient homogenizing 7 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with paclitaxel vesicle type phospholipid gel through the deep bid lyophilization (-70 ℃ of pre-freezes 2 hours, then freezing 8 hours at-40 ℃, sublimed up into 25 ℃ through 24 hours again, at last 28 ℃ of dryings 2 hours), add water for injection again and be uniformly dispersed to 10L, the packing of 10ml/ bottle namely gets paclitaxel vesicle type phospholipid injection.
The preparation of embodiment 3 paclitaxel vesicle type phospholipid gel injections
Prescription: (1000 bottles)
Preparation technology
(1) under nitrogen protection, the volume ratio that paclitaxel 150g, two myristoyl PHOSPHATIDYL ETHANOLAMINE 7500g and stigmasterol 3000g is dissolved in water for injection, ethanol and the tert-butyl alcohol is in the 12000ml solution of 1:2:7, slow magnetic agitation is 30 minutes under 45 ℃, obtains taxusol-lipid solution;
(2) with above-mentioned solution under-60 ℃ of conditions freezing 8 hours, be warming up to 25 ℃ with 5 ℃/hour speed again, 25 ℃ of dryings 2 hours, obtain forming loose spongiform drying solid at last;
(3) under nitrogen protection, above-mentioned solid dispersion is contained in the aqueous solution of trehalose 1500g in 20000ml, ultrasonic 15 minutes of 600W, 200bar does the gradient homogenizing 6 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with paclitaxel vesicle type phospholipid gel through the deep bid lyophilization (-70 ℃ of pre-freezes 3 hours, then freezing 6 hours at-50 ℃, sublimed up into 20 ℃ through 18 hours again, at last 25 ℃ of dryings 3 hours), add water for injection again and be uniformly dispersed to 25L, the 25ml/ packing namely gets paclitaxel vesicle type phospholipid injection.
The preparation of embodiment 4 paclitaxel vesicle type phospholipid gel injections
Prescription: (1000 bottles)
Preparation technology
(1) under nitrogen protection, the volume ratio that paclitaxel 30g, two myristoyl PHOSPHATIDYL ETHANOLAMINE 1650g and stigmasterol 600g is dissolved in water for injection, ethanol and the tert-butyl alcohol is in the 3000ml solution of 1:2:7, slow magnetic agitation is 20 minutes under 45 ℃, obtains taxusol-lipid solution;
(2) with above-mentioned solution under-60 ℃ of conditions freezing 6 hours, be warming up to 25 ℃ with 6 ℃/hour speed again, 25 ℃ of dryings 4 hours, obtain forming loose spongiform drying solid at last;
(3) under nitrogen protection, above-mentioned solid dispersion is contained in the aqueous solution of trehalose 300g in 4000ml, ultrasonic 25 minutes of 500W, 200bar does the gradient homogenizing 7 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4)-65 ℃ of pre-freezes 3 hours, then-50 ℃ freezing 8 hours, sublimed up into 25 ℃ through 20 hours again, 30 ℃ of dryings 2 hours, with 1000 bottles of lyophilized powder packing, obtain paclitaxel vesicle type phospholipid gel freezing-dried powder injection at last.
The preparation of Comparative Examples 1-4 paclitaxel vesicle type phospholipid gel injection
Adopt respectively with embodiment 1-4 in identical production technology, the composition in will the Comparative Examples 1-4 as shown in following table 1 is made paclitaxel vesicle type phospholipid gel injection respectively:
Used composition among the table 1 Comparative Examples 1-4
Wherein, "/" expression is not used.
The investigation of test example 1 vesicle type phospholipid gel freeze-dried powder form
With the vesicle type phospholipid gel sample for preparing among embodiment 1-4 and the Comparative Examples 1-4 at freeze etching transmission electron microscope (FFTEM, can be with reference to Brandl, Chem.Phys.Lipids in 1997 such as M, 87,65) carry out quality investigation, mainly carry out morphologic observation and the particle size determination of sample.The results are shown in the following table 2.
The investigation result of table 2 vesicle type phospholipid gel and liposome
As shown in Table 2, gained paclitaxel vesicle type phospholipid gel form rule among the embodiment of the invention 1-4, size is even; And gained paclitaxel vesicle type phospholipid gel form is irregular among the Comparative Examples 1-4, and particle diameter is inhomogeneous.
Particularly, even when adopting same production technology, the paclitaxel vesicle type phospholipid gel that the particle appearance of gained paclitaxel vesicle type phospholipid gel, particle diameter obviously are better than gained among the Comparative Examples 1-4 respectively among the embodiment 1-4.This shows that the quality of gained paclitaxel vesicle type phospholipid gel obviously is inferior to the present invention when the composition beyond the used composition of use the present invention.Experiment in addition shows, when the composition consumption is outside the composition amount ranges that the present invention limits, can not obtain the uniform paclitaxel vesicle of gel form rule size type phospholipid gel.
After the vesicle type phospholipid gel sample for preparing among embodiment 1-4 and the Comparative Examples 1-4 being used the water for injection dissolving of 100ml, 200ml, 500ml and 100ml respectively, jolting, paclitaxel vesicle type phospholipid gel can be transformed into the paclitaxel small unilamellar vesicle after the aquation, detect the liposome particle diameter with photon correlation spectroscopy Nicomp370, the results are shown in following table 3.
Table 3 liposome particle diameter testing result
As shown in Table 4, can be transformed into superior in quality paclitaxel small unilamellar vesicle after the vesicle type phospholipid gel preparation aquation of embodiment 1-4 preparation, can not form superior in quality paclitaxel small unilamellar vesicle after the vesicle type phospholipid gel preparation aquation of Comparative Examples 1-4, also prove absolutely the superiority of the present invention's combination.
The mensuration of test example 2 envelop rates
With the rotating speed high speed centrifugation of the paclitaxel vesicle type phospholipid gel injection for preparing among embodiment 1-4 and the Comparative Examples 1-4 (lyophilized powder of embodiment 4 and Comparative Examples 4 namely gets paclitaxel vesicle type phospholipid gel injection after adding the water for injection dissolving of 5ml) with 5000r/min, centrifugal 20 minutes, get supernatant, use the methanol ultrasonic dissolution, the HPLC method is surveyed content of taxol, the computational envelope rate the results are shown in the following table 4.
Table 4 entrapment efficiency determination result
| Numbering | Embodiment 1 | Comparative Examples 1 | Embodiment 2 | Comparative Examples 2 | Embodiment 3 | Comparative Examples 3 | |
Comparative Examples 4 |
| Envelop rate | 95.3% | 82.7% | 96.6% | 81.6% | 95.9% | 82.4% | 96.2% | 81.0% |
As shown in Table 4, the envelop rate of the vesicle type phospholipid gel preparation of embodiment 1-4 preparation is higher than the envelop rate of the vesicle type phospholipid gel preparation of Comparative Examples 1-4 significantly.When the composition beyond using the used composition of the present invention was described, perhaps when the composition consumption was outside the composition amount ranges that the present invention limits, the vesicle type phospholipid gel envelop rate of gained vesicle type phospholipid gel was lower than the present invention.
Test example 3 study on the stability
With the sample of embodiment of the invention 1-4 preparation and the injection paclitaxel (lot number: 20101211 of listing, Yangzijiang Pharmaceutical Group Co., Ltd, production address: No. 1, South Road, Taizhou City Yangtze River, Jiangsu Province) place following 6 months of the condition of 40 ℃ of high temperature, relative humidity 75% respectively, carry out accelerated test and investigate, experimental result is shown in the following table 5.
Table 5 accelerated test result
As shown in Table 5, when accelerating June, the listing formulation content reduces, and related substance raises; And sample character of the present invention, content and related substance variation are all not obvious, illustrate that product stability of the present invention is good.
The test of test example 4 percolation ratios
Get the sample of test routine 1-4 and Comparative Examples 1-4 preparation, at ambient temperature, respectively at 0 day, 30 days, 60 days, 90 days and 180 days, make regular check on, measure envelop rate, with the dose of sealing in 0 day relatively, calculate percolation ratio, the results are shown in the following table 4.
Table 6 percolation ratio result of the test
As shown in Table 6, during long term storage, the paclitaxel vesicle type phospholipid gel injection percolation ratio for preparing among the embodiment of the invention 1-4 changes little, and the injection percolation ratio for preparing among the Comparative Examples 1-4 increases gradually, vesicle type phospholipid gel seepage is serious, and the paclitaxel vesicle type phospholipid gel injection of this explanation the present invention preparation has higher stability.
The mensuration of test example 5 blood drug level
63 rats are divided into 9 groups at random, every group of injection for preparing among drug administration by injection embodiment 1-4 and the Comparative Examples 1-4 respectively, and commercially available (lot number: 20101211, Yangzijiang Pharmaceutical Group Co., Ltd, production address: No. 1, South Road, Taizhou City Yangtze River, Jiangsu Province), specification is the 5ml:30mg paclitaxel injection, and the administration of every rat paclitaxel is 30mg.Respectively at 0.5h, 1h, 1.5h, 2h, 3h, 6h, 8h, 12h and 24h, take a blood sample after the administration, blood sample is measured blood drug level with the HPLC-MS method after treatment.The paclitaxel vesicle type phospholipid gel injection for preparing among the paclitaxel vesicle type phospholipid gel injection for preparing among the drafting embodiment 1-4, the Comparative Examples 1-4 and blood drug level and the time relation curve of commercially available injection paclitaxel are shown in the accompanying drawing 1.
As shown in Figure 1, the paclitaxel vesicle type phospholipid gel injection for preparing among embodiment 1-4 and the Comparative Examples 1-4 is compared with commercially available paclitaxel injection, the paclitaxel vesicle type phospholipid gel injection for preparing among the embodiment of the invention 1-4 has the slow release slow release effect, has improved bioavailability.More than experiment has proved absolutely the superiority of the present invention's combination, has obtained the remarkable technique effect of unexpected excellence.
Industrial applicibility
More than by the specific embodiment the present invention is further specified: according to the result of embodiment and Comparative Examples as can be known, paclitaxel vesicle type phospholipid gel of the present invention has the good surface appearance character, stability is high, percolation ratio is low, the time of staying in vivo is long, the bioavailability height has the favorable industrial using value.
Below through the specific embodiment and the embodiment the present invention is had been described in detail; but should understand; these explanations do not constitute any restriction to scope of the present invention; in the case of without departing from the spirit and scope of protection of the present invention; can carry out multiple modification, improvement and replacement to technical solutions and their implementation methods of the present invention, because these all fall within the scope of protection of the present invention.
Claims (8)
1. paclitaxel vesicle type phospholipid gel injection is characterized in that being made by the composition of following weight proportion:
1 part of paclitaxel
Two myristoyl PHOSPHATIDYL ETHANOLAMINE 40-70 parts
Stigmasterol 10-30 part
Trehalose 5-20 part,
Weight ratio between wherein said stigmasterol and the two myristoyl PHOSPHATIDYL ETHANOLAMINE is 1:3-2:5, and the ratio between paclitaxel and the two myristoyl PHOSPHATIDYL ETHANOLAMINE is 1:50-1:60;
And its preparation method comprises the steps:
(1) under nitrogen protection, paclitaxel, two myristoyl PHOSPHATIDYL ETHANOLAMINE and stigmasterol are dissolved in the proper amount of solvent, 45 ℃ were descended slow magnetic agitation 15-30 minute, and obtained taxusol-lipid solution;
(2) with above-mentioned solution lyophilization, complete until drying, obtain forming loose spongiform drying solid;
(3) under nitrogen protection with above-mentioned solid dispersion in aqueous trehalose, ultrasonic 15～30 minutes of 400W-600W, 200bar does the gradient homogenizing 6～8 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with the lyophilization of paclitaxel vesicle type phospholipid gel, the lyophilized powder direct packaging obtains paclitaxel vesicle type phospholipid gel freezing-dried powder injection; Perhaps with the lyophilization of paclitaxel vesicle type phospholipid gel, add the back packing that is uniformly dispersed of a certain amount of water for injection then, namely get paclitaxel vesicle type phospholipid gel injection.
2. paclitaxel vesicle type phospholipid gel injection according to claim 1 is characterized in that being made by the composition of following weight proportion:
1 part of paclitaxel
Two myristoyl PHOSPHATIDYL ETHANOLAMINE 50-60 parts
20 parts of stigmasterol
10 parts of trehaloses.
3. according to each described paclitaxel vesicle type phospholipid gel injection among the claim 1-2, it is characterized in that specification is 5ml:30mg, 10ml:60mg or 25ml:150mg.
4. a method for preparing each described paclitaxel vesicle type phospholipid gel injection among the claim 1-3 is characterized in that comprising the steps:
(1) under nitrogen protection, paclitaxel, two myristoyl PHOSPHATIDYL ETHANOLAMINE and stigmasterol are dissolved in the proper amount of solvent, 45 ℃ were descended slow magnetic agitation 15-30 minute, and obtained taxusol-lipid solution;
(2) with above-mentioned solution lyophilization, complete until drying, obtain forming loose spongiform drying solid;
(3) under nitrogen protection with above-mentioned solid dispersion in aqueous trehalose, ultrasonic 15～30 minutes of 400W-600W, 200bar does the gradient homogenizing 6～8 times to 900bar in high pressure homogenizer then, obtains the paclitaxel vesicle type phospholipid gel of even pasty state;
(4) with the lyophilization of paclitaxel vesicle type phospholipid gel, the lyophilized powder direct packaging obtains paclitaxel vesicle type phospholipid gel freezing-dried powder injection; Perhaps with the lyophilization of paclitaxel vesicle type phospholipid gel, add the back packing that is uniformly dispersed of a certain amount of water for injection then, namely get paclitaxel vesicle type phospholipid gel injection.
5. the method for preparing paclitaxel vesicle type phospholipid gel injection according to claim 4, wherein, the solvent described in the step (1) is the mixed solvent of water for injection, ethanol and the tert-butyl alcohol.
6. the method for preparing paclitaxel vesicle type phospholipid gel injection according to claim 5, wherein said mixed solvent is that the volume ratio of water for injection, ethanol and the tert-butyl alcohol is the solution of 1:2:7.
7. the method for preparing paclitaxel vesicle type phospholipid gel injection according to claim 4, it is characterized in that cryodesiccated process is described in the step (2): under-60 ℃ of conditions freezing 5-8 hour, be warming up to 25 ℃ with 5-10 ℃/hour speed again, at last at 25 ℃ of dry 2-4 hours to fully dry.
8. the method for preparing paclitaxel vesicle type phospholipid gel injection according to claim 4, it is characterized in that cryodesiccated process is described in the step (4) :-60 ℃～-70 ℃ pre-freezes 2～3 hours, then freezing 6～8 hours at 40 ℃～-50 ℃, sublimed up into 20 ℃～25 ℃ through 18～24 hours again, extremely drying was complete in 1～3 hour 25 ℃～30 ℃ dryings at last.
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| CN101011357A (en) * | 2006-11-16 | 2007-08-08 | 西安力邦医药科技有限责任公司 | Process for preparing Paclitaxel liposome preparation |
| CN101190214A (en) * | 2007-01-31 | 2008-06-04 | 广东庆发药业有限公司 | Paclitaxel injection and preparation method thereof |
| CN101584663A (en) * | 2008-05-22 | 2009-11-25 | 广州瑞济生物技术有限公司 | Novel delivery system of Duoxitasai lipidosome for injection and preparation method thereof |
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| CN101011357A (en) * | 2006-11-16 | 2007-08-08 | 西安力邦医药科技有限责任公司 | Process for preparing Paclitaxel liposome preparation |
| CN101190214A (en) * | 2007-01-31 | 2008-06-04 | 广东庆发药业有限公司 | Paclitaxel injection and preparation method thereof |
| CN101584663A (en) * | 2008-05-22 | 2009-11-25 | 广州瑞济生物技术有限公司 | Novel delivery system of Duoxitasai lipidosome for injection and preparation method thereof |
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