CN100508972C - Mutamycine C multivesicular liposome and preparing method thereof - Google Patents
Mutamycine C multivesicular liposome and preparing method thereof Download PDFInfo
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- CN100508972C CN100508972C CNB2006100251594A CN200610025159A CN100508972C CN 100508972 C CN100508972 C CN 100508972C CN B2006100251594 A CNB2006100251594 A CN B2006100251594A CN 200610025159 A CN200610025159 A CN 200610025159A CN 100508972 C CN100508972 C CN 100508972C
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Abstract
A multilocular liposome of mitomycin for preventing and treating tumor is prepared through dissolving the lipoid component (neutral phosphatide, cholesterol and neutral lipid) in organic solvent, dissolving mitomycin in buffering salt solution, mixing, emulsifying to obtain water-in-oil primary emulsion, adding external water phase containing isotonic regulator, stirring to become water-in-oil-in-water emulsion, and removing organic solvent.
Description
Technical field
The present invention relates to a kind of slow releasing preparation of ametycin, particularly a kind of Mutamycine C multivesicular liposome and preparation method thereof.
Background technology
(Mitomycin C is the antibiotics broad-spectrum anti-cancer drug that people such as Hata found in nineteen fifty-five MMC) to ametycin, is cell cycle nonspecific agent (CCNSA).Ametycin to the G1 phase of tumor cell, particularly late G1 phase early the S phase the most responsive, behind enzyme activation, its effect is similar to difunctional or three function alkylating agents, can cross link take place with DNA in tissue, it is synthetic to suppress DNA, to RNA and albumen are synthetic certain inhibitory action is arranged also.Up to the present, this product has been used more than 20 year clinically, is the active drug of the various solid tumors of treatment.It is mainly used in alimentary tract cancer clinically, and pulmonary carcinoma, breast carcinoma, cervical cancer etc. are also had significant curative effect, also can be used for the therapeutic alliance of malignant lymphoma.In addition, ametycin also can be used for trabeculectomy treatment glaucoma, because of behind the resection operation normal take place bulbar conjunctiva down and between the sclera fibroblast hyperplasia, fibrosis, cicatrization cause and filter passage and block, make to filter to steep and be difficult to form, incidence rate can reach 15%～30%, and the antimetabolite ametycin can increase the success rate of trabeculectomy, has obtained affirming of clinical application for many years.Ametycin is a kind of as cancer therapy drug, and serious general toxicity is arranged, and the treatment window is narrow, and the character instability of medicine own can only be made freeze-dried powder at present, faces with before being mixed with the solution intravenous injection.Ametycin is the half-life weak point in vivo, needs the multiple injection administration in the therapeutic process, need have the nursing staff to nurse after the administration, very inconvenience, patient's poor compliance.
On the other hand, people such as Kim find and have studied many bubbles (many capsules) liposome at first in nineteen eighty-three, they pass through multi-emulsion method, with phospholipid, cholesterol and neutral lipid is filmogen, with chloroform and ether is organic solvent, successfully prepared multivesicular liposomes, and the microstructure of multivesicular liposomes that relied on optical microscope and electron microscopic observation.In preparation process, at first drug solution and oil phase are formed W/O (Water-In-Oil) type colostrum under mechanicals efforts, again with colostrum and outer water are mixed must emulsion, dry up by nitrogen and to remove organic solvent and make the multivesicular liposomes suspension.The particulate mean diameter of multivesicular liposomes is between 5～50 μ m.Be prepared into multivesicular liposomes by multi-emulsion method and have high envelop rate (20%～90%), high drug load, high stability (can preserve more than a year under 4 ℃), low seepage, slow releasing function (can postpone to discharge a couple of days) to several weeks.Because of its non-concentrically ringed topological structure, make multivesicular liposomes form medicine " bank " in the injection site, along with the continuous metabolism of phospholipid bilayer, the medicine that is encapsulated in the vesicle progressively is released into blood or diseased region, and performance well postpones release action.By regulating parameter and the prescription ratio in the preparation process, easily the control drug release time is between several days to several weeks.
Yet, the problem that exists is at present: because different active constituents of medicine has the different physicochemical properties and the mechanism of action, its preparation envelop rate and drug release and preparation stability have bigger difference, therefore at concrete medicine and release request thereof, need research to adopt specific prescription and technology.
Summary of the invention
The technical problem to be solved in the present invention is above-mentioned problem, and a kind of Mutamycine C multivesicular liposome with slow releasing function and preparation method thereof is provided.
Technical scheme of the present invention comprises: a kind of preparation method of Mutamycine C multivesicular liposome, it can comprise the following steps:
1. lipid components is dissolved in the organic solvent, with as the lipid phase, wherein this lipid components comprises that weight ratio is neutral phospholipid and the cholesterol of 2:1～4:1, and to account for molar percentage in lipid components be 2～6% neutral lipid, and this neutrality phospholipid is 20～40mg/ml in the concentration of lipid in mutually;
2. ametycin is dissolved in that to make its concentration in the buffer salt solution be 20 μ g/ml～3000 μ g/ml, regulates pH and be 7.4～9.2 with as interior water;
3. isopyknic described interior water is added lipid phase upper strata, mixing and emulsifying makes the water-in-oil type colostrum;
4. the upper strata that the outer water of 2～6 times of volumes is added described water-in-oil type colostrum is stirred and is formed W/O/W (W/O/W) type emulsion, and its China and foreign countries' aqueous phase contains the osmotic pressure instrumentality of 3.2～6g/100ml;
5. remove the organic solvent in the emulsion that 4. step obtain and make Mutamycine C multivesicular liposome.
Wherein, above-mentioned neutral phospholipid and cholesterol are filmogen; Cholesterol is a membrane stabilizer, can regulate the flowability of liposome rete by changing the phospholipid phase transition temperature, thereby improve the stability of liposome, reduces the drug leakage that causes because of the phase transformation of liposome rete in the storage process.The cholesterol consumption is too small, and its regulating action is very little, is difficult to form the phospholipid bilayer of liposome when consumption is excessive, studies show that the liposome that forms when phospholipid and cholesterol weight ratio are between 2:1～4:1 is the most stable.
The neutral phospholipid of step of the present invention described in 1. is meant that net charge when being in its isoelectric point, IP is zero phospholipid, and it is selected from the natural and synthetic phospholipid such as lecithin, soybean phospholipid, cephalin, sphingomyelins, hydrogenated soya phosphatide, dimyristoyl phosphatidyl choline (DMPC), dioleoyl phospholipid phatidylcholine (DOPC), PHOSPHATIDYL ETHANOLAMINE, DOPE (DOPE) one or more; The present invention is preferably from lecithin, dioleoyl phospholipid phatidylcholine and DOPE.
And the said neutral lipid of the present invention is meant and itself does not form the vesicle ability, and lack the oil of charged or hydrophilic " head " group or fatty, as in glycerol trioleate, trilaurin, decanoin, tricaprylin, tricaproin, vitamin E and the Squalene one or more.It mainly plays the support effect in liposome, be distributed in non-concentric aqueous chamber phospholipid bilayer node place, supports the topological structure of many bubble (many capsules) liposomees; Neutral lipid is one of deciding factor that forms many bubble (many capsules) liposomees.The preferred glycerol trioleate of the present invention.
This organic solvent can be any solvent of energy lipin dissolving composition, is selected from ether, chloroform, dichloromethane, isopropyl ether, oxolane, halogen ether and the halogen ester one or more usually.Preferred dichloromethane of the present invention or E-C mixture.Be more preferred from dichloromethane, because of dichloromethane can effectively dissolve phospholipid and lipoid, and toxicity is lower than chloroform, and it is more excellent to volatilize speed.
The lipoid that can also contain electronegative or positive charge in this step lipid components 1., electrically charged lipoid can effectively be regulated the surface of liposome electric charge, makes the electronegative or positive electricity of liposome, to strengthen preparation stability.The weight ratio of this lipoid and neutral phospholipid is 1:5～1:60, and the lipoid consumption acts on not obvious very little, then increases cost too much, the preferred 1:5～1:20 of the present invention.
Electronegative lipoid of the present invention is selected from Phosphatidylserine, two palmityl Phosphatidylserine (DPPS), distearyl Phosphatidylserine (DSPS), phosphatidyl glycerol, two palmityl phosphatidyl glycerols (DPPG), distearyl phosphatidyl glycerol (DSPG), phosphatidylinositols, two palmityl phosphatidylinositols (DPPZ), distearyl phosphatidylinositols (DSPZ), phosphatidic acid, in two palmityl phosphatidic acid (DPPA) and the G 12S3P (DSPA) one or more; And described positively charged lipoid is selected from diacyl trimethylamine propane, diacyl dimethylamine propane, stearylamine (SA) and the collagen protein one or more.
Lipoid of the present invention is positively charged lipoid such as stearylamine and/or collagen protein most preferably.Because ametycin is electronegativity under alkali condition, makes the phospholipid bilayer oppositely charged help improving the envelop rate of electronegativity medicine, and can reduce drug leakage.In addition, add certain electric charge lipoid and can increase the aqueous chamber, thereby increase envelope volume, and can increase the stability of Liposomal formulation, reduce particle precipitation and gathering.
Consider and contain assorted 3 important component parts of three propane of amino benzoquinone, carbamic acid quinone and ammonia in the molecular structure of ametycin, all unstable under acid, alkali, illumination, hot conditions, three-membered ring generation acid catalysis open loop in acid solution, and alkalescence is crossed the hydrolysis of strong mephenesin Carbamate chain; The catabolite inefficacy of ametycin in addition, and toxicity is bigger.So in order to strengthen the stability of ametycin, the inventor find after deliberation above-mentioned steps 2. in, aqueous pH values can reduce the degraded of ametycin to certain alkaline range in regulating, then ametycin is the most stable in 8.0～8.7 scopes for pH.
Preferably, the concentration of step interior aqueous phase ametycin 2. is 500～1000 μ g/ml, in this scope, can have higher drug level, can obtain higher entrapment again.The buffer salt solution of step described in 2. then selected in the liposome preparation field phosphate buffered solution of conventional use for use.
Step interior aqueous phase 2. can also add and is no more than 10% (w/v), generally is no more than the osmotic pressure instrumentality of 4% (w/v), with further enhancing preparation stability; Wherein, reach hereinafter mentioned bulking value (w/v) percentage ratio herein and all refer to g/100ml.
Osmotic pressure instrumentality of the present invention can be the material that is usually used in regulating osmotic pressure in the prior art, as carbohydrates such as glucose, sucrose, mannitol, maltose, and sodium chloride etc.Preferred glucose of the present invention and/or sucrose.
Above-mentioned steps 3. described in mixing and emulsifying can adopt prior art, as handling by apparatus and method such as stirring, vibration, sonication, ultrasound wave, high speed shear refiners; The present invention preferably adopts the high speed shear refiner.
And above-mentioned steps mix to form emulsion and the step removal solvent method in 5. in 4. and all can adopt prior art, remove wherein that solvent method includes but are not limited to that air-flow dries up, reduction vaporization etc., usually select for use gases such as noble gases such as nitrogen, helium, argon and hydrogen, carbon dioxide to dry up solvent, the most frequently used and cost is low is that nitrogen dries up.
The present invention also provides a kind of Mutamycine C multivesicular liposome, and it is made by above-mentioned preparation method.
The present invention can solve medicine stability and general toxicity two aspect problems by ametycin being made slow release " bank " preparation.Ametycin is encapsulated in the liposome preserves the degraded that can effectively reduce medicine under 4 ℃ of conditions; Medicament slow release can make local drug concentration maintain effective treatment concentration, reduces administration number of times, improves patient's compliance.Mutamycine C multivesicular liposome of the present invention has high envelop rate, in vivo, all show the good slow release effect in the outer test, thereby existing preparation can have better cell proliferation and antitumous effect, and reduced medication number of times and total dosage thus, further reduced general toxicity.
Description of drawings
Fig. 1 is the release in vitro-time plot of Mutamycine C multivesicular liposome of the present invention.
Fig. 2 is that Mutamycine C multivesicular liposome suspension of the present invention is at the intravital blood drug level-time plot of mice.
Fig. 3 is that in contrast existing mitomycin c solution is at the intravital blood drug level-time plot of mice.
The specific embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
The Mutamycine C multivesicular liposome particle diameter adopts the granule size analyser to detect among the following embodiment; The entrapment efficiency determination method is:
Get 0.5mL Mutamycine C multivesicular liposome suspension of the present invention, add the 4.5mL normal saline, evenly mixed, centrifugal 5 minutes of 600 * g, separation of supernatant and precipitation.Detect free ametycin concentration in (2005 editions two p176 of Chinese Pharmacopoeia) supernatant according to the analytical method of having set up; With the Triton X-100 solution rupture of membranes dissolving that precipitates with 10%, measure the concentration of the ametycin of sealing in accordance with the law, calculate the envelop rate of Mutamycine C multivesicular liposome with following formula:
Ametycin total amount * 100 in envelop rate (%)=entrapped ametycin amount/suspension
Step 1: precision takes by weighing lecithin 200mg, cholesterol 50mg, stearylamine 40mg and glycerol trioleate 17.4mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 5mg ametycin and sucrose 100mg, uses an amount of dissolved in distilled water, and regulates pH to 8.0 with sodium dihydrogen phosphate and sodium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with high speed shear refiner (Fluko, ATS industrial system company limited) step 2 gained mixture is acted on 9 minutes under the rotating speed of 10000rpm, make the w/o type colostrum;
Step 4: for making dichloromethane microsphere suspension, the outer water that 20mL is contained 3.2% (w/v) glucose is added in the upper strata of w/o type colostrum, with 15 seconds of rotating speed effect of 4500rpm, forms the dichloromethane microsphere suspension;
Step 5: for making multivesicular liposomes, above-mentioned suspension is injected the 1000mL conical flask that fills the outer water of 30mL, pass to nitrogen (8L/min), dichloromethane was slowly removed in 37 ℃ of water-baths in 20 minutes; In conical flask, add the 60mL normal saline, under 600 * g condition, separated liposome in centrifugal 5 minutes, abandon supernatant,, gained is precipitated redispersion in 5～10ml normal saline, get the multivesicular liposomes suspension with normal saline washing precipitation three times.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 87.4%.
Step 1: precision takes by weighing lecithin 200mg, cholesterol 50mg, collagen protein 10mg and glycerol trioleate 15.6mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 5mg ametycin and sucrose 200mg, uses an amount of dissolved in distilled water, and regulates pH to 8.7 with potassium dihydrogen phosphate and potassium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: for making dichloromethane microsphere suspension, the outer water that 20mL is contained 6.0% (w/v) glucose is added in the upper strata of w/o type colostrum, with 15 seconds of rotating speed effect of 4500rpm, forms the dichloromethane microsphere suspension;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 75.1%.
Step 1: precision takes by weighing dioleoyl phospholipid phatidylcholine 100mg, cholesterol 50mg, stearylamine 20mg and glycerol trioleate 3.4mg, with 5ml chloroform ether mixture (volume ratio is 1:1) dissolving, as the lipid phase;
Step 2: precision takes by weighing the 2.5mg ametycin, uses an amount of dissolved in distilled water, and regulates pH to 8.4 with sodium hydrogen phosphate and sodium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: for making chloroform-ether microsphere suspension, the outer water that 60mL is contained 3.2% (w/v) glucose is added in the upper strata of w/o type colostrum, with 15 seconds of rotating speed effect of 4500rpm, forms chloroform-ether microsphere suspension;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 80.6%.
Step 1: precision takes by weighing DOPE 150mg, cholesterol 50mg, collagen protein 15mg and glycerol trioleate 7.5mg, with 5ml chloroform ether mixture (volume ratio is 1:1) dissolving, as the lipid phase;
Step 2: precision takes by weighing the 3.75mg ametycin, and 100mg sucrose is used an amount of dissolved in distilled water, and regulates pH to 6.5 with sodium hydrogen phosphate and sodium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: for making chloroform-ether microsphere suspension, the outer water that 40mL is contained 5.7% (w/v) glucose is added in the upper strata of w/o type colostrum, with 15 seconds of rotating speed effect of 4500rpm, forms chloroform-ether microsphere suspension;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 76.8%.
Step 1: precision takes by weighing lecithin 200mg, cholesterol 100mg, stearylamine 40mg and glycerol trioleate 20.4mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 5mg ametycin and sucrose 100mg, uses an amount of dissolved in distilled water, and regulates pH to 9.2 with sodium hydrogen phosphate and sodium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: for making dichloromethane microsphere suspension, 20mL is contained the upper strata that the outer water of 3.2% (w/v) glucose is added in the w/o type colostrum,, form the dichloromethane microsphere suspension with 15 seconds of rotating speed effect of 4500rpm;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 81.7%.
Step 1: precision takes by weighing lecithin 120mg, cholesterol 30mg and glycerol trioleate 1.6mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 5mg ametycin and glucose 200mg, uses an amount of dissolved in distilled water, and regulates pH to 8.0 with dipotassium hydrogen phosphate and potassium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 67.4%.
Step 1: precision takes by weighing lecithin 200mg, cholesterol 100mg, phosphatidyl glycerol 40mg and glycerol trioleate 20.4mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 5mg ametycin and sucrose 100mg, uses an amount of dissolved in distilled water, and regulates pH to 8.0 with dipotassium hydrogen phosphate and potassium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: for making dichloromethane microsphere suspension, 20mL is contained the upper strata that 4% (w/v) sucrose and the arginic outer water of 40mM are added in the w/o type colostrum,, form the dichloromethane microsphere suspension with 15 seconds of rotating speed effect of 4500rpm;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 54.9%.
Step 1: precision takes by weighing lecithin 200mg, cholesterol 50mg, stearylamine 40mg and glycerol trioleate 17.4mg, with the dissolving of 5ml dichloromethane, as the lipid phase;
Step 2: precision takes by weighing 15mg ametycin and sucrose 100mg, uses an amount of dissolved in distilled water, and regulates pH to 8.0 with sodium dihydrogen phosphate and sodium hydroxide, is settled to 5ml, as interior water, slowly adds lipid phase upper strata;
Step 3: with embodiment 1;
Step 4: with embodiment 1;
Step 5: with embodiment 1.
Recording the Mutamycine C multivesicular liposome particle diameter is 5～50 μ m; Envelop rate is 79.4%.
Used lecithin is the goldschmidt chemical corporation product in the foregoing description; Collagen protein, dioleoyl phospholipid phatidylcholine, DOPE, phosphatidyl glycerol are purchased the company in Sigma; Stearylamine is purchased the company in Fluka; Cholesterol is purchased in last sea blue season development in science and technology company limited; Glycerol trioleate is purchased in Guangzhou Chemical Reagent Factory; All the other reagent are conventional commercially available prod.
Experimental example 1 Mutamycine C multivesicular liposome stability study
1. sample preparation
1.1. according to three batches of Mutamycine C multivesicular liposome suspensions of embodiment 1 preparation, lot number is Y5M3-M1, Y5M3-M2, Y5M3-M3;
1.2. according to embodiment 1, but interior aqueous phase does not use buffer salt solution to regulate pH value, only prepares three batches of Mutamycine C multivesicular liposome suspensions with dissolved in distilled water, standardize solution, lot number is Y5M3-N1, Y5M3-N2, Y5M3-N3, with in contrast.
2. preserve and sampling
| 1st month | 2nd month | 3rd month | |
| Y5M3—M1 | 0.21％ | 0.25％ | 0.31％ |
| Y5M3—M2 | 0.25％ | 0.24％ | 0.29％ |
| Y5M3—M3 | 0.18％ | 0.25％ | 0.38％ |
| Y5M3—N1 | 1.59％ | 3.44％ | 4.87％ |
| Y5M3—N2 | 2.01％ | 3.67％ | 5.09％ |
| Y5M3—N3 | 1.64％ | 3.20％ | 4.73％ |
The result shows, in regulating Mutamycine C multivesicular liposome in water pH, can significantly improve preparation stability, content decline in three months only is about 0.3%.
The release in vitro of experimental example 2 Mutamycine C multivesicular liposomes is measured
Get the Mutamycine C multivesicular liposome suspension 10ml of embodiment 1, dilute with 40ml phosphate buffer solution (pH8.0), the gained suspension is placed 37 ℃ of constant temperature shaking tables (rotating speed is 15rpm), take out the sample of equivalent at preset time point, with 600 * g centrifugalize supernatant and precipitation, measure the amount of ametycin in the supernatant, the drug release percentage ratio of each time point calculates with following formula in accordance with the law:
Ametycin original vol * 100 of sealing in the amount/multivesicular liposomes of free ametycin in drug release percentage ratio (%)=supernatant
The result shows that drug release has reached more than 80% when the 6th day (144 hours), and the ametycin of sealing discharges (as shown in Figure 1) fully substantially.The multivesicular liposomes that ametycin of the present invention is described has tangible slow release effect.
The mice interior medicine dynamics research of experimental example 3 Mutamycine C multivesicular liposomes
By the pharmacokinetic study of injecting in the mouse muscle, prove the sustained release profile in vivo test effect of Mutamycine C multivesicular liposome.
Get the multivesicular liposomes that embodiment 1 makes, preparation ametycin concentration is that the multivesicular liposomes suspension of 10mg/mL is standby.
1. animal
(male and female half and half, body weight are 18～22g) to 80 of Kunming mouses, raise, manage mice according to " management of laboratory animal regulation ".Overnight fasting before on-test, and do the carotid artery intubate.
2. administration and blood specimen collection
80 mices are divided into two groups at random, inject the Mutamycine C multivesicular liposome suspension and the mitomycin c solution (contrast) of 2.5mg/kg dosage respectively at right leg muscle.After the administration, by carotid artery intubate blood sample collection (0.3mL), inject isopyknic normal saline to mice after taking a blood sample at preset time point at every turn.The centrifugal blood sample of 10,000 * g is 10 minutes immediately, and the gained mice plasma in-20 ℃ of freezing preservations, is treated further analyzing and testing.
3. sample detection
3.1. the foundation of analytical method
The accurate blood plasma 0.5ml that draws places the 5ml plastic centrifuge tube, the accurate methanol solution (1000 μ g/ml) that adds mark contrast nitrobenzaldehyde in the 100 μ l, add the 3.0ml ethyl acetate, vortex extraction 5min, centrifugal (11,000rpm/min, 10min) upper organic phase is transferred to other-the 5ml centrifuge tube in, lower floor adds the 2.0ml ethyl acetate again and carries out the extraction second time, and the combined ethyl acetate layer dries up in 35～38 ℃ of water-bath nitrogen.Residue redissolves with 200 μ l methanol, and mixing is centrifugal, gets supernatant 20 μ l and carries out HPLC and analyze, and the record chromatogram carries out quantitative analysis with the ratio of sample peak and interior mark peak area.
3.2. sample detection
The ametycin amount of each time point sample of assay determination utilizes Topfit 2.0 programs to calculate blood drug level-time relation data.
4. result of the test
As shown in Figure 2, Mutamycine C multivesicular liposome of the present invention is tangible slow release behavior in the mice body, and the concentration of ametycin is still about 1 μ g/ml in the detected blood in 5 days (120 hours) back; And mitomycin c solution in contrast (not sealing) after the intramuscular injection about 4 hours blood drug level just be reduced to below the 0.1 μ g/ml (as shown in Figure 3).Therefore can prove that ametycin has effectively been realized interior slow release of body of ametycin after multivesicular liposomes is sealed.
Claims (10)
1, a kind of preparation method of Mutamycine C multivesicular liposome, it comprises the following steps:
1. lipid components is dissolved in the organic solvent, with as the lipid phase, wherein this lipid components comprises that weight ratio is neutral phospholipid and the cholesterol of 2:1～4:1, and to account for molar percentage in lipid components be 2～6% neutral lipid, and this neutrality phospholipid is 20～40mg/ml in the concentration of lipid in mutually;
2. ametycin is dissolved in pH and is in 7.4～9.2 the buffer salt solution, making its concentration is 20 μ g/ml～3000 μ g/ml, with as interior water.
3. isopyknic described interior water is added lipid phase upper strata, mixing and emulsifying makes the water-in-oil type colostrum;
4. the upper strata that the outer water of 2～6 times of volumes is added described water-in-oil type colostrum mixes forming W/O/W type emulsion, and its China and foreign countries' aqueous phase contains the osmotic pressure instrumentality of 3.2～6g/100ml;
5. remove the organic solvent in the emulsion that 4. step obtain and make Mutamycine C multivesicular liposome.
2, preparation method as claimed in claim 1 is characterized in that also containing in the lipid components described in this step 1. the lipoid of electronegative or positive charge, and the weight ratio of this lipoid and neutral phospholipid is 1:5～20.
3, preparation method as claimed in claim 2 is characterized in that described electronegative lipoid is selected from one or more in Phosphatidylserine, two palmityl Phosphatidylserine, distearyl Phosphatidylserine, phosphatidyl glycerol, two palmityl phosphatidyl glycerols, distearyl phosphatidyl glycerol, phosphatidylinositols, two palmityl phosphatidylinositols, distearyl phosphatidylinositols, phosphatidic acid, two palmityl phosphatidic acid and the G 12S3P; Described positively charged lipoid is selected from one or more in diacyl trimethylamine propane, diacyl dimethylamine propane, stearylamine and the collagen protein.
4, preparation method as claimed in claim 3 is characterized in that this lipoid is stearylamine and/or collagen protein.
5, preparation method as claimed in claim 1 is characterized in that neutral phospholipid during step 1. is selected from one or more in lecithin, soybean phospholipid, hydrogenated soya phosphatide, dimyristoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine, PHOSPHATIDYL ETHANOLAMINE, DOPE, cephalin, the sphingomyelins; This neutral lipid is selected from one or more in glycerol trioleate, trilaurin, decanoin, tricaprylin, tricaproin, vitamin E and the Squalene; This organic solvent is selected from one or more in ether, chloroform, dichloromethane, isopropyl ether, oxolane, halogen ether and the halogen ester.
6, preparation method as claimed in claim 5, it is characterized in that this neutrality phospholipid is selected from lecithin, dioleoyl phospholipid phatidylcholine and DOPE, this neutral lipid is a glycerol trioleate, and this organic solvent is dichloromethane or E-C mixture.
7, preparation method as claimed in claim 1 is characterized in that during step 2. ametycin is dissolved in pH and is in 8.0～8.7 the phosphate buffered solution, and making its concentration is 500～1000 μ g/ml, with as interior water.
8, preparation method as claimed in claim 1 is characterized in that the interior aqueous phase described in step is 2. can also add〉0g/100ml, and≤the osmotic pressure instrumentality of 4g/100ml.
9,, it is characterized in that described osmotic pressure instrumentality is glucose and/or sucrose as claim 1 or 8 described preparation methoies.
10, the Mutamycine C multivesicular liposome that makes as each described preparation method of claim 1～8.
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| CN101536981B (en) * | 2008-03-19 | 2010-11-17 | 上海医药工业研究院 | Clonidine hydrochloride multivesicular liposome and preparation method thereof |
| CN104706595B (en) * | 2013-12-16 | 2018-01-23 | 厦门市壳聚糖生物科技有限公司 | A kind of cancer target mitomycin C lipoid plastid and preparation method thereof |
| CN106924747A (en) * | 2017-03-17 | 2017-07-07 | 临沂大学 | Imitative lipoprotein structure pharmaceutical carrier of a kind of nanometer and its preparation method and application |
-
2006
- 2006-03-28 CN CNB2006100251594A patent/CN100508972C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
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| 水溶性药物8-氯腺苷多囊脂质体的制备. 严文伟，肖超菊，齐宪荣，王文浩.北京大学学报(医学版)，第34卷第4期. 2002 |
| 水溶性药物8-氯腺苷多囊脂质体的制备. 严文伟，肖超菊，齐宪荣，王文浩.北京大学学报(医学版)，第34卷第4期. 2002 * |
| 顺铂缓释多囊脂质体的制备和体外释放性能研究. 肖超菊，齐宪荣，艾尼瓦尔，魏树礼.药学学报，第38卷第2期. 2003 |
| 顺铂缓释多囊脂质体的制备和体外释放性能研究. 肖超菊，齐宪荣，艾尼瓦尔，魏树礼.药学学报，第38卷第2期. 2003 * |
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| CN101045049A (en) | 2007-10-03 |
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