CN1965820A - Lipid microsphere, lyophilized lipid microsphere containing docetaxel and preparation process thereof - Google Patents
Lipid microsphere, lyophilized lipid microsphere containing docetaxel and preparation process thereof Download PDFInfo
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- CN1965820A CN1965820A CNA2006101547105A CN200610154710A CN1965820A CN 1965820 A CN1965820 A CN 1965820A CN A2006101547105 A CNA2006101547105 A CN A2006101547105A CN 200610154710 A CN200610154710 A CN 200610154710A CN 1965820 A CN1965820 A CN 1965820A
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Abstract
The invention relates to a liposome micro-sphere agent with Doxitasa, and dry liposome micro-sphere, wherein the mass density of Doxitasa is 1-40mg/ml; the liposome micro-sphere component comprises Doxitasa, oil component, emulsifier, and injection water, to be prepared into vein injection. The invention also provides a relative Doxitasa dry liposome micro-sphere agent, with high load, high packing rate, better stability, and little side effect, which can be stored and transmitted easily, with target property. The inventive agent supports batch production.
Description
Technical field
The invention belongs to the pharmaceutical preparations technology field, specifically, the present invention relates to a kind of lipid microsphere, lyophilizing lipid microsphere and preparation method that contains docetaxel.
Background technology
Docetaxel (Docetaxel/Taxotere, Docetaxel) by the French (RhonePoulencRorer of Rhone-Poulenc Rorer, i.e. Sanofi-Aventis company now) develops, successively go on the market in succession at present in the world more or less a hundred countries that comprise states such as China and U.S., day.
Docetaxel is a kind of in the bearing taxanes, and it can suppress the tumor cell differentiation by " freezing " the inherent skeleton of cell in fact.Cytoskeleton is made up of microtubule, and microtubule in cell cycle assembling and partition phenomenon can take place in good time.Docetaxel can promote the microtubule assembling and stop the microtubule partition, suppress the tumor cell differentiation thus, finally cause death of neoplastic cells (Fulton B.Spencer CM.Docetaxel:A review of its pharmacodynamic and pharmacokineticproperties and therapeutic efficacy in the management of metastatic breast cancer.Drugs, 1996:51 (6): 1075).
Docetaxel has at present obtained to be used for the treatment of now in each major country of the whole world that three kinds of the most common tumors of the world are the multiple indication of breast carcinoma, nonsmall-cell lung cancer and carcinoma of prostate, and has become one of the most frequently used or standard treatment of these cancer kinds treatments or.Docetaxel and and also carrying out widely as breast carcinoma of early stage neoadjuvant and treatment gastric cancer, tumor of head and neck, the esophageal carcinoma, ovarian cancer etc. with scheme.In March, 2006, the approval docetaxel of U.S. FDA was used for the treatment of late gastric cancer.Clinical pharmacology studies confirm that the anti-tumor activity of docetaxel is better than paclitaxel (in vitro study shows that its antitumor action is 1.9～9.3 times of paclitaxel), and does not have cross resistance with paclitaxel.Clinical random research in recent years shows, treats medicine as a linearize of treatment epithelial ovarian cancer, and it is identical that the treatment curative effect that docetaxel adds carboplatin and paclitaxel add carboplatin, adds carboplatin but its neurotoxicity is starkly lower than paclitaxel.(Burger R A，Disaia P J，Roberts J A，et al.Phase II trial ofvinorelbine in recurrent and progressive epithelial ovarian cancer[J].Gynecol Oncol，1999，72(2)：148-153.Ray coquard I.Docetaxel and ovarian cancerEJ].Bull Cancer，2004，91(2)：159-165.)
The docetaxel molecular formula is: C
43H
53NO
14
Structural formula is as follows:
Docetaxel belongs to bearing taxanes, and its independent medication oral administration biaavailability is 8% only, and being made into ejection preparation is to improve one of its bioavailability means.But the extremely low about 1 μ g/ml of the dissolubility of docetaxel in water has seriously limited its use clinically.
The docetaxel injection preparation of clinical practice at present has freeze-dried powder and small-volume injection.The Docetaxel for Injection of clinical usefulness is the injection concentrated solution, be 2 cillin bottle packings, wherein one bottle is the tween solution of docetaxel, another bottle is an ethanol water, and clinical practice is very inconvenient, must instil at the dilution posterior vein, earlier ethanol water is injected the tween solution of docetaxel during clinical practice, fully rock, observe just can be applied to the patient after clear and bright after 5 minutes, and may produce secondary pollution because of medicine dissolution or dilution; Because docetaxel is water insoluble, the unstability after the dilution might be separated out crystallization after the preparation dilution, and exosmoses when transfusion, can cause local red and swollen, sclerosis.
In addition, docetaxel injecta also exists bigger untoward reaction and toxic and side effects in clinical practice.The docetaxel main adverse reactions shows as angioedema, anaphylaxis, dermal toxicity reaction, the vascular exosmosis regional cutaneous necrosis that causes exosmosing.Blood vessel and local tissue necrosis are one of main adverse reactions in the docetaxel clinical application.
At Chinese patent " a kind of lipomul that contains docetaxel and preparation method thereof " (application number: 200510084055.6, at on 07 18th, 2005 applying date, open day: on December 21st, 2005) in the docetaxel fat breast is disclosed, its quality volumetric concentration scope that contains docetaxel is 0.1～1.0mg/ml, and specification is 1～500mg.Docetaxel is clinical to be 100mg/m with the consumption
2, with body surface area 1.5m
2Calculate, then its volumes of formulation is 150～1500ml.Because its concentration is lower, cause volumes of formulation bigger, be not convenient for patient's medication, the needs that can not satisfy transportation simultaneously and store.
Lipid microsphere agent (Lipid Microsphere, LM), it is the newtype drug targeted therapeutic carrier, the normal method that adopts is the lipid core part that pharmaceutical pack is wrapped in lipid microsphere, because this structure also is similar to microsphere, be a kind of be soft substrate and the microsome disperse system sealed by interfacial film such as immobilized artificial membrane with oil composition.
Summary of the invention
The present invention seeks to overcome the prior art deficiency, solve that docetaxel instability, dissolubility are extremely low, erious adverse reaction and make the technical problem of patient compliance difference in the medication, it is low also to solve simultaneously in the prior art concentration of docetaxel injecta, the problem of the inconvenient and storing inconvenience of the medication in clinical.
The invention provides a kind of docetaxel high drug load lipid microsphere preparation that intravenous injection is used that is used for.Its mass concentration scope is 1～40mg/ml.
Docetaxel lipid microsphere provided by the present invention comprises active component docetaxel, oiliness composition, emulsifying agent and water for injection.
Wherein the quality volumetric concentration of oiliness composition is 50～500mg/ml; The quality volumetric concentration of emulsifying agent is 5～300mg/ml.
Wherein the oiliness composition is selected from injection soybean oil, safflower oil, medium chain triglycerides, long-chain fatty acid ester, one or more in fractionated oil or modified oil, olive oil, cetylate, Oleum Cocois, Oleum sesami, fish oil, the vitamin E; In the oiliness compositions such as preferred soybean oil, medium chain triglycerides, vitamin E one or more, most preferably vitamin E.
Emulsifying agent is one or more in soybean phospholipid, lecithin, poloxamer, tween 80, Polyethylene Glycol, cholesterol, oleic acid, benzyl alcohol, ethyl lactate, ethyl oleate, benzyl benzoate, arabic gum, Saponin and the agar.In preferred soybean phospholipid, lecithin, poloxamer, the oleic acid one or more, most preferably lecithin.In emulsifying agent, having quite a few can also can treat as simultaneously helps Emulsion to use, to strengthen the emulsifying film strength, prevent that emulsion droplet from merging, as among poloxamer, Polyethylene Glycol, benzyl alcohol, ethyl lactate, oleic acid, ethyl oleate, benzyl benzoate, arabic gum, Saponin or the agar one or more.
The present invention can also add suitable stabilizing agent, such as in anhydrous sodium sulfite, VC, nitrogen, dibenzylatiooluene, EDTA and disodium, alpha-tocopherol, α-tocopheryl acetate, the hydroquinone etc. one or more.
The present invention can also add isoosmotic adjusting agent and rise simultaneously and help Stabilization, isoosmotic adjusting agent to be selected from glycerol, mannitol, xylitol, sorbitol, glucose, the sucrose etc. one or more.
Docetaxel lipid microsphere provided by the present invention is made up of following component:
Docetaxel 1--40g
Stabilizing agent 0--200g
Osmolyte regulator 0--200g
Water for injection adds to 1000ml
The present invention also provides a kind of docetaxel lipid microsphere preparation method, comprises the following steps:
(1) preparation oil phase: oiliness composition, emulsifying agent, docetaxel are joined in the oiliness composition preparing tank, stir oil phase;
(2) preparation water: with water soluble ingredient, add in the water preparing tank, stir, dissolving gets water;
(3) prepare thick emulsion: oil-phase solution is joined aqueous phase, stirring, adding water for injection to overall solution volume is ormal weight, regulates pH value to 4～8.5, continues to stir, and gets thick emulsion;
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, and homogenizing is until reaching suitable granularity requirements;
(5) degerming, fill;
Temperature when wherein said preparation oil water phase, thick emulsion is 20～80 ℃.
Specifically, preparation method of the present invention is:
(1) preparation oil phase: precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel is added in phospholipid and the injection oiliness composition, attemperation and pH are stirred to whole dissolvings, get oil phase;
(2) preparation water: with water soluble ingredients such as waters for injection, add in the water preparing tank, stir, dissolving remains on 20～60 ℃ with solution temperature, gets water;
(3) prepare thick emulsion: will join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 20～80 ℃, stirred 5～60 minutes, adding water for injection to overall solution volume then is ormal weight, regulate pH value to 4～8.5 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion.
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 20～60 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements.
(5) degerming: adopt canned preceding aseptic filtration or canned back moist heat sterilization.Aseptic filtration, with behind the homogenizing the aseptic filtration of microporous filter membrane of employing 0.2; 120 ℃ of flowing steam sterilizations are 15 minutes after the fill.Fill: according to required specification, under the condition of logical nitrogen, packing promptly obtains docetaxel lipid microsphere of the present invention.
The present invention also provides a kind of docetaxel lyophilization dry lipid microsphere, forms by following component:
Docetaxel 1--40g
Stabilizing agent 0--200g
Osmolyte regulator 0--200g
Frozen-dried supporting agent 0--400g
Freeze drying protectant 0--200g
Water for injection adds to 1000ml
Many adjuvants are arranged; as mannitol, xylitol, sorbitol, glucose, sucrose; can use as Osmolyte regulator, stabilizing agent, frozen-dried supporting agent and freeze drying protectant simultaneously; so when selecting some Osmolyte regulator or stabilizing agent for use; can only add very a spot of frozen-dried supporting agent and/or freeze drying protectant, even not add.
Wherein injection oiliness composition is selected from injection soybean oil, safflower oil, medium chain triglycerides, long-chain fatty acid ester, one or more in fractionated oil or modified oil, olive oil, cetylate, Oleum Cocois, Oleum sesami, fish oil, the vitamin E; In the oiliness compositions such as preferred soybean oil, medium chain triglycerides, vitamin E one or more, most preferably vitamin E.
Emulsifying agent is one or more in soybean phospholipid, lecithin, poloxamer, tween 80, Polyethylene Glycol, cholesterol, oleic acid, benzyl alcohol, ethyl lactate, ethyl oleate, benzyl benzoate, arabic gum, Saponin and the agar.In preferred soybean phospholipid, lecithin, poloxamer, the oleic acid one or more, most preferably lecithin.In emulsifying agent, having quite a few can also can treat as simultaneously helps Emulsion to use, to strengthen the emulsifying film strength, prevent that emulsion droplet from merging, as among poloxamer, Polyethylene Glycol, benzyl alcohol, ethyl lactate, oleic acid, ethyl oleate, benzyl benzoate, arabic gum, Saponin or the agar one or more.
The present invention can also add suitable stabilizing agent, such as in anhydrous sodium sulfite, VC, nitrogen, dibenzylatiooluene, EDTA and disodium, alpha-tocopherol, α-tocopheryl acetate, the hydroquinone etc. one or more.
The present invention can also add isoosmotic adjusting agent and rise simultaneously and help Stabilization, isoosmotic adjusting agent to be selected from glycerol, mannitol, xylitol, sorbitol, glucose, the sucrose etc. one or more.
Wherein said proppant and freeze drying protectant are one or more mixture in mannitol, xylitol, sorbitol, glucose, sucrose, glycine, sodium chloride, lactose, citric acid, the hydrolyzed protein, preferred mannitol and glycine.
The present invention also provides a kind of docetaxel lipid microsphere preparation method, comprises the following steps:
(1) preparation oil phase: oiliness composition, emulsifying agent, docetaxel are joined in the oiliness composition preparing tank, attemperation, stir oil phase;
(2) preparation water: with water soluble ingredient, add in the water preparing tank, stir, dissolving gets water;
(3) prepare thick emulsion: oil-phase solution is joined aqueous phase, stirring, adding water for injection to overall solution volume is ormal weight, regulates pH value to 4～8.5, continues to stir, and gets thick emulsion;
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, and homogenizing is until reaching suitable granularity requirements;
(5) degerming adds proppant, freeze drying protectant, lyophilization, fill;
Temperature when wherein said preparation oil phase, water, thick emulsion is 20～80 ℃.
Specifically, preparation method of the present invention is:
(1) preparation oil phase: precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel is added in phospholipid and the injection oiliness composition, attemperation and pH are stirred to whole dissolvings, get oil phase;
(2) preparation water: with water soluble ingredients such as waters for injection, add in the water preparing tank, stir, dissolving remains on 20～60 ℃ with solution temperature, gets water;
(3) prepare thick emulsion: will join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 20～80 ℃, stirred 5～60 minutes, adding water for injection to overall solution volume then is ormal weight, regulate pH value to 4～8.5 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion.
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 20～60 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements.
(5) degerming: adopt canned preceding aseptic filtration or canned back moist heat sterilization.Aseptic filtration, with behind the homogenizing the aseptic filtration of microporous filter membrane of employing 0.2; 120 ℃ of flowing steam sterilizations are 15 minutes after the fill, add proppant, freeze drying protectant, lyophilization, and fill: according to required specification, under the condition of logical nitrogen, packing promptly obtains docetaxel lipid microsphere of the present invention.
Wherein said proppant, freeze drying protectant also can add in the water preparation.
Docetaxel lipid microsphere provided by the invention or lyophilizing docetaxel lipid microsphere have the following advantages:
1. improved the dissolubility of docetaxel in ejection preparation breakthroughly, making had only the quality volumetric concentration to have only 0.1--1.0mg/ml to rise to 1--40mg/ml originally, thereby make things convenient for clinical practice to a great extent, the loaded down with trivial details bulky deficiency of docetaxel fat breast of also having avoided when both having avoided the clinical use of normal injection and common lyophilized formulations.With the docetaxel consumption is 100mg/m
2Body surface area 1.5m
2Calculate, its volumes of formulation of docetaxel fat breast is 150～1500ml, and calculates with the intermediate concentration 20mg/ml among concentration range 1～40mg/ml of the present invention, only needs 7.5ml, overcome because be difficult to import the fat milk of so a large amount of volumes, and failed to reach the disadvantage of treatment concentration.The present invention had both made things convenient for patient's use, and also convenient simultaneously the storage transported.
2, improve physics and chemical stability greatly, be difficult for separating out and oxidation, avoided that related substance exceeds standard easily in storage, shortcomings such as the not enough and partial crystallization of the solution clarity that occurs easily when using clinically.
Because docetaxel is wrapped in the lipid microsphere preparation, physics and chemical property are stable to be improved, and is difficult for separating out and oxidation.Constant product quality has solved using and do not store the crystallize phenomenon can occur, and its storage period is more than 2 years.Especially be prepared into the Docetaxel for Injection lipid microsphere through lyophilization, its stability will be improved greatly.
3, reduced toxicity and untoward reaction is few, alleviated zest, increased safety of clinical administration, and improved the advantages such as compliance of patient's medication blood vessel.Easily cause allergic reaction because of a large amount of tween 80s that use in the prior art, can not contain or only contain the tween 80 of minute quantity in the component of docetaxel lipid microsphere disclosed in this invention, thereby the anaphylaxis that can reduce tween 80 and produce has increased the safety of medicine; Docetaxel is wrapped in the lipid microsphere preparation, gives full play to the superiority of dosage form, has avoided contacting with the direct of blood vessel, has alleviated the zest to blood vessel, has avoided untoward reaction such as docetaxel medication process medium vessels and local tissue necrosis.
4, this preparation has local targeting.Lipid microsphere has targeting distribution characteristics in the body, has both reduced the drug level in blood and the hetero-organization thereof, has improved curative effect again.Because the microgranule of certain particle diameter can concentrate in reticuloendothelial system abundant liver and pulmonary with blood circulation after injection enters human body, therefore particle diameter has the guiding distribution performance of relative targeting to the reticular tissue cell system at the medicine carrying lipid microsphere of this scope, this for the curative effect that improves cancer therapy drug, reduce that it is highly beneficial to Normocellular infringement, can lower the untoward reaction of general further, increase the useful effect of medicine.Emulsifying agent kind, charge, particle diameter etc. are all influential to target organ and targeting.
5, medicine can reach slow release, keeps drug effect.Lipid microsphere energy prolong drug circulation time in vivo, the long period keeps drug effect.The factor that influences the release of lipid microsphere Chinese medicine comprises: kind, viscosity, interfacial film and the emulsion droplet granularity etc. of pharmaceutical properties (as dissolubility, partition coefficient, molecular size, ionization constant etc.), pH value, oil composition emulsifying agent and additive.
6, preparation method provided by the present invention is to realize the big production of scale, the preparation technology of stable processing technique, controllable parameters, favorable reproducibility.
Through drug inspection, docetaxel lipid microsphere provided by the invention and the every index of lyophilizing docetaxel lipid microsphere all meet drug standard.
Description of drawings
The particle size distribution figure of Fig. 1 embodiment 1
The particle size distribution figure of Fig. 2 embodiment 2
The assay chromatogram of Fig. 3 embodiment 1
The related substance chromatogram of Fig. 4 embodiment 1
The specific embodiment
Embodiment 1
Prescription is formed:
Docetaxel 10g
VE 100g
Phosphatidase 12 5g
Oleic acid 50g
PEG400 25g
Sucrose 50g
EDTA disodium 0.05g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, with docetaxel, PEG400, oleic acid, VE, phospholipid, join in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 50 ℃, oil phase; Sucrose and EDTA disodium, a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 50 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 45 ℃, stir and sheared 15 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, packing promptly obtains the docetaxel lipid microsphere.
It is as follows to make the finished product physicochemical characteristics according to present embodiment:
[finished product character]: the even emulsion of milky;
[particle size distribution]: with 5% glucose as retarder thinner, after the clinical dosage dilution, laser diffraction particle analyzer Nicomt-380, U.S. Patrticle Sizing Systems (PSS) company] sample cell in, measure size, mean diameter is 103.6nm, and particle size distribution figure sees Fig. 1.
[hydration dispersibility]: respectively solvent for injection as 5% glucose injection in, disperse in 30 seconds, to disperse to form clear solution voluntarily through jolting.
[hydration stability]: get this product and add 5% glucose injection.Measure medicament contg and size respectively.To place 2,4 in room temperature respectively, and after 6,12,18,24 hours, measure mean diameter, with comparison in 0 o'clock, particle diameter increased less than 5%.Behind the mistake 0.22 μ m filter membrane of placing after 24 hours, measure medicament contg, with 24 hours before relatively, should be 99.57% (n=3) of former dose.
[assay]
Measure according to high performance liquid chromatography (2000 editions two appendix VD of Chinese Pharmacopoeia), liquid-phase condition is as follows:
Tianjin, island high-efficient liquid phase analysis instrument
Chromatographic column: Diamonsil C
18(200mm * 4.6mm * 5um);
Mobile phase: methanol-0.02mol/L sodium acetate solution (regulating pH value 5.0) (75: 25) with acetic acid
Detect wavelength: 232nm;
Column temperature: 30 ℃; Flow velocity: 1ml/min; Sample size: 20ul.
[related substance inspection]
Measure according to high performance liquid chromatography (2000 editions two appendix V D of Chinese Pharmacopoeia), liquid-phase condition is as follows:
Tianjin, island high-efficient liquid phase analysis instrument
Chromatographic column: Diamonsil C
18(200mm * 4.6mm * 5um);
Mobile phase: methanol-0.02mol/L sodium acetate solution (with acetic acid adjust pH 5.0) (65: 35)
Detect wavelength: 232nm;
Column temperature: 30 ℃; Flow velocity: 1ml/min; Sample size: 20ul.
[entrapment efficiency determination]:
Lipid microsphere preparation of Chinese medicine content assaying method: lipid microsphere is handled the back analyze according to condition sample introduction in [assay] item;
The entrapment efficiency determination method:
Getting this product adds water and makes the suspension that drug level is 1mg/mL.
The mensuration (W1) of total dose: precision pipettes the 0.5mL preparation in the 25mL volumetric flask, adds the dehydrated alcohol breakdown of emulsion, gets settled solution and is diluted to scale, records total dose (W1) by the medicine assay method.
Be encapsulated in the dose (W2) in the lipid microsphere: pipette the 1mL lipid microsphere in microcentrifugal tube, the centrifugal 10min of 1500r/min, precision pipettes the 0.5mL supernatant in the 25mL volumetric flask, add the dehydrated alcohol breakdown of emulsion, get settled solution and be diluted to scale, record total dose (W2) by the medicine assay method.
Envelop rate calculates: W2/W1 * 100%
Result of the test: the envelop rate of the prepared docetaxel lipid microsphere of present embodiment agent is 99.21 ± 0.71% (n=3)
[hemolytic test]:
One, test material
Medicine: the docetaxel lipid microsphere that present embodiment makes; Ai Su (docetaxel enriched oil injection, Hengrui Medicine Co., Ltd., Jiangsu Prov.).Being diluted to 7.0mg/ml with 5% glucose injection before the administration tests.
Animal: Japanese white big ear rabbit
Two, test method
2% red cell suspension preparation: select healthy rabbits, get fresh blood, stir blood with Glass rod and make into defibrinated blood to remove to defibrinate, change in the graduated centrifuge tube then, reuse normal saline flushing three times adds the 10ml normal saline at every turn, and is centrifugal behind the mixing, remove supernatant, till supernatant does not show redness.Press the erythrocytic volume of gained then, be mixed with 2% suspension, be for experiment with normal saline.
Get 12 in test tube, the according to the form below proportional quantity adds various solution successively, behind the mixing, places 30 minutes in 37 ℃ of calorstats, adds not commensurability medicinal liquid then respectively.After shaking up, put in 37 ℃ of calorstats and observed 4 hours, observed once every 15 minutes in 1 hour.2-4 hour, observed the primary first-order equation result every 1 hour, judge whether haemolysis by following standard, the clear and bright redness of haemolysis (+) solution, it is residual that the pipe end does not have erythrocyte.Haemolysis (-) erythrocyte does not all sink, the supernatant achromatism and clarity.Blood coagulation:, after jolting, can not disperse though red blood cell condensation appears in haemolysis not.Generally with 0.3ml injection (the 3rd pipe) but the person that do not produce the haemolysis in 2 hours thinks injection.Phenomenon if any red blood cell condensation, can further judge it is true cohesion or pseudo agglutination by purgation, if condensation product again can homodisperse after the test tube jolting or condensation product is placed on the microscope slide, drip 2 normal saline at the coverslip edge, examining under a microscope the cohesion erythrocyte can be pseudo agglutination by the person of breaking up, and test sample can supply clinical practice; If condensation product is not shaken diffusing or be not true cohesion by the person of breaking up on slide, test sample should not be for clinical use.
Table hemolytic result of the test (7mg/ml)
| Sample | The test tube numbering |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| He matches lipid microsphere Ai Su 2% red cell suspension normal saline distilled water many two | 2.5 2.0 0 0.5 0 | 2.5 2.1 0 0.4 0 | 2.5 2.2 0 0.3 0 | 2.5 2.3 0 0.2 0 | 2.5 2.4 0 0.1 0 | 2.5 2.5 0 0 0 | 2.5 0 2.5 0 0 | 2.5 2.0 0 0 0.5 | 2.5 2.1 0 0 0.4 | 2.5 2.2 0 0 0.5 | 2.5 2.3 0 0 0.2 | 2.5 2.5 0 0 0.1 |
Annotate: the solution unit that each pipe adds is ml.The negative not haemolysis control tube of the 6th pipe, the positive complete hemolysis control tube of the 7th pipe.
Three, result of the test and conclusion
The docetaxel lipid microsphere is when 7mg/ml concentration, can not produce haemolysis at test tube 0.1～0.4, during more than or equal to 0.5ml, long-time placement can produce utmost point slight hemolysis, Ai Su can not produce haemolysis when 0.1～0.2ml, during more than or equal to 0.3ml, long-time placement can produce utmost point slight hemolysis, can produce haemolysis more than the placement 50min during 0.5ml.Docetaxel lipid microsphere hemolytic is less than listing enriched oil injection.
[blood vessel irritation test]:
One, test material
Medicine: the docetaxel lipid microsphere that present embodiment makes; Ai Su (docetaxel enriched oil injection, Hengrui Medicine Co., Ltd., Jiangsu Prov.).
Dosage design: the administration volume press 10ml/kg, and administration is preceding, and to be mixed with concentration with 5% glucose injection be 1.8mg/ml (be equivalent to be grown up each consumption 3 times).
Animal: Japan large ear rabbit, body weight 2.0-3.0kg, female, hero half and half
Two, test method
Select 18 of healthy rabbits, body weight 2.0-3.0kg, female, hero half and half is divided into 3 groups at random, and 6 every group, first group is matched group, the intravenous injection normal saline, second group is intravenous injection docetaxel lipid microsphere, the 3rd group of positive control drug group.The administration volume is all by 10ml/kg, successive administration 3 days, once a day, last was injected back 24 hours, and the blood vessel of first perusal injection site and surrounding tissue have or not the variation of hyperemia, edema, degeneration and necrosis etc., get 3 rabbit for every group then, by proximal part, cut the rabbit ear (containing blood vessel) 1.5-3cm with the injection site, fix with 10% formalin, histopathologic examination is carried out in the conventional organization section.Observe injection site rabbit ear edge vein blood vessel and have or not vasodilation hyperemia, blood stasis, vessel wall inflammation, necrosis, changes such as endothelial cell is loose, come off, blood vessel surrounding tissue inflammatory cell infiltration.In addition 3 rabbit are behind the last intravenous administration 7 days, with the blood vessel of method perusal injection site and the variation of surrounding tissue, and carry out histopathologic examination.
Three, result of the test
After naked eyes and pathological tissue are checked rabbit vein injection lipid microsphere and normal saline, do not find the irritant reaction relevant with medicine, finding changes system's injection puncture mechanical irritation reaction.Listing docetaxel enriched oil injection can cause that with same concentration and dosed administration the rabbit ear light-moderate vascular stimulation occurs and reacts.
[study on the stability]
Docetaxel lipid microsphere that present embodiment is made and commercially available injection are placed on the stability (the results are shown in following table) of investigating character, content, related substance and granularity under 30 ℃ of accelerated test conditions and the 4 ℃ of long-term storage conditions.
The result shows: what present embodiment made compares with listing water for injection injection, more stable.The study on the stability result of the test that embodiment 1 makes shows: docetaxel lipid microsphere outward appearance, detection pH value, breast grain and free fatty are all qualified, show in 2 years, to place and stablize, illustrate that the docetaxel lipid microsphere that present embodiment makes has advantages of higher stability.
Docetaxel lipid microsphere lyophilized formulations is that solid state exists, stability is better than the docetaxel lipid microsphere of liquid condition, the docetaxel lipid microsphere has had good stable, so but docetaxel lipid microsphere lyophilized formulations thinks also have good stable.
The study on the stability result
| Time (moon) | Related substance (%) the present invention/Comparative formulation | Content (%) the present invention/Comparative formulation |
| 0 | 0.12/0.23 | 98.53/97.59 |
| 1 | 0.16/0.29 | 98.12/96.42 |
| 2 | 0.22/0.34 | 96.89/92.27 |
| 3 | 0.25/0.47 | 95.53/89.92 |
| 6 | 0.25/0.57 | 93.43/86.45 |
| 9 | 0.30/0.65 | 92.24/83.53 |
Embodiment 2
Prescription is formed:
Docetaxel 10g
VE 100g
Phosphatidase 12 5g
Oleic acid 50g
PEG400 25g
Sucrose 50g
EDTA disodium 0.05g
Mannitol 100g
Glycine 20g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, phospholipid, PEG400, oleic acid, VE are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 50 ℃, gets oil phase; Sucrose and EDTA disodium, a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 50 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 45 ℃, stir and sheared 15 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane, add the mannitol and the glycine of recipe quantity; Under the condition of logical nitrogen, packing ,-40 ℃ of following pre-freezes, lyophilization promptly obtains docetaxel lyophilization dry lipid microsphere of the present invention.
The docetaxel lyophilization dry lipid microsphere that makes according to present embodiment is white or little yellow lyophilizing block or powder.
Prescription is formed:
Docetaxel 2g
VE 80g
Phosphatidase 12 0g
Polyoxyethylene castor oil 30g
Water for injection adds to 1000ml
Preparation method is identical with embodiment 1, makes the docetaxel lipid microsphere.
Embodiment 4
Prescription is formed:
Docetaxel 5g
VE 80g
Phosphatidase 12 0g
Poloxamer 188 40g
PEG400 20g
Mannitol 40g
Water for injection adds to 1000ml
Preparation method is identical with embodiment 1, makes the docetaxel lipid microsphere.
Prescription is formed:
Docetaxel 5g
Soybean oil 50g
Medium chain triglycerides 50g
Poloxamer 188 20g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, soybean oil, medium chain triglycerides, tween 80 are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 45 ℃, gets oil phase; Poloxamer 188 and a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 45 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 40 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6.2 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.45 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, canned, place 15 minutes dresses of 120 ℃ of flowing steam sterilizations of rotating type sterilization cabinet promptly to obtain the docetaxel lipid microsphere.
Embodiment 6
Prescription is formed:
Docetaxel 10g
Soybean oil 50g
Medium chain triglycerides 50g
Oleic acid 20g
Poloxamer 188 20g
Lactose 100g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, soybean oil, medium chain triglycerides, oleic acid are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 50 ℃, gets oil phase; With the poloxamer 188 and the lactose of recipe quantity, and in a certain amount of water for injection adding water preparing tank, stir, dissolving remains on 50 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 45 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.22 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, fill ,-40 ℃ of following pre-freezes, lyophilization, docetaxel lyophilization dry lipid microsphere in getting.
Embodiment 7
Prescription is formed:
Docetaxel 10g
Soybean oil 60g
Medium chain triglycerides 60g
Poloxamer 188 20g
Mannitol 40g
Water for injection adds to 1000ml
Other step promptly obtains the docetaxel lipid microsphere with embodiment 1.
Embodiment 8
Prescription is formed:
Press group component and prepare raw material, adjuvant and other components
Docetaxel 15g
Soybean oil 60g
Medium chain triglycerides 60g
Oleic acid 60g
Poloxamer 188 50g
Lactose 120g
Water for injection adds to 1000ml
Preparation method is identical with embodiment 6, makes the docetaxel lyophilization dry lipid microsphere.
Embodiment 9
Prescription is formed:
Docetaxel 35g
Oleum Gossypii semen 100g
Phosphatidase 12 5g
Oleic acid 50g
Benzyl benzoate 0.15g
Glycerol 20g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, phospholipid, Oleum Gossypii semen, benzyl benzoate, oleic acid are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 45 ℃, gets oil phase; Glycerol and a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 45 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 40 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, packing promptly obtains the docetaxel lipid microsphere.
Prescription is formed:
Docetaxel 18g
Fish oil 80g
Phosphatidase 12 5g
Oleic acid 50g
Glucose 120g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, phospholipid, oleic acid, fish oil are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 40 ℃, gets oil phase; Glucose sugar and a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 40 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 40 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6.2 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, packing ,-40 ℃ of following pre-freezes, lyophilization promptly obtains the docetaxel lyophilization dry lipid microsphere.
Embodiment 11
Prescription is formed:
Docetaxel 12g
Medium chain fatty acid ester 100g
Phosphatidase 12 5g
PEG400 25g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, phospholipid, PEG400, medium chain fatty acid ester, VE are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 45 ℃, gets oil phase; Sucrose and a certain amount of water for injection of recipe quantity are added in the water preparing tank, stir, dissolving remains on 45 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 40 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, packing promptly obtains the docetaxel lipid microsphere.
Embodiment 12
Prescription is formed:
Docetaxel 10g
Olive oil 100g
Phosphatidase 12 5g
Polyoxyethylene castor oil 50g
EDTA disodium 0.05g
Water for injection adds to 1000ml
Precision takes by weighing supplementary material; Under the condition of nitrogen protection, docetaxel, phospholipid, polyoxyethylene castor oil are joined in the oiliness composition preparing tank, be stirred to whole dissolvings, solution temperature remains on 50 ℃, gets oil phase; The EDTA disodium of recipe quantity, a certain amount of water for injection are added in the water preparing tank, stir, dissolving remains on 50 ℃ with solution temperature, must be water; To join in the aqueous phase solution in the high-speed shearing equipment, oil-phase solution is joined lentamente the aqueous phase that is stirring, keeping temperature is 45 ℃, stir and sheared 20 minutes, add the ormal weight cumulative volume of water for injection then to solution, regulate pH value to 6 with sodium hydroxide or hydrochloric acid solution solution, continue to stir 15 minutes, get thick emulsion; Thick emulsion is joined in the high pressure homogenize equipment, regulate suitable pressure, keep 30 ℃ of temperature, homogenizing is 2～6 times repeatedly, until reaching suitable granularity requirements; Adopt 0.2 the aseptic filtration of microporous filter membrane; Under the condition of logical nitrogen, packing promptly obtains the docetaxel lipid microsphere.
Claims (10)
1. docetaxel lipid microsphere agent, the mass concentration scope that it is characterized in that docetaxel is 1～40mg/ml.
2. docetaxel lipid microsphere according to claim 1 comprises active component docetaxel, oiliness composition, emulsifying agent and water for injection.
3. docetaxel lipid microsphere according to claim 2 is characterized in that the quality volumetric concentration of oiliness composition in the described lipid microsphere is 50～500mg/ml; The quality volumetric concentration of emulsifying agent is 5～300mg/ml.
4. docetaxel lipid microsphere according to claim 2, it is characterized in that the oiliness composition is selected from injection soybean oil, safflower oil, medium chain triglycerides, long-chain fatty acid ester, one or more in fractionated oil or modified oil, olive oil, cetylate, Oleum Cocois, Oleum sesami, fish oil, the vitamin E; Emulsifying agent is one or more in soybean phospholipid, lecithin, poloxamer, tween 80, Polyethylene Glycol, cholesterol, oleic acid, benzyl alcohol, ethyl lactate, ethyl oleate, benzyl benzoate, arabic gum, Saponin and the agar.
5. docetaxel lipid microsphere according to claim 2 is characterized in that containing in the component stabilizing agent, isoosmotic adjusting agent.
6. the preparation method of a docetaxel lipid microsphere as claimed in claim 1 comprises the following steps:
(1) preparation oil phase: oiliness composition, emulsifying agent, docetaxel are joined in the oiliness composition preparing tank, attemperation, stir oil phase;
(2) preparation water: with water soluble ingredient, add in the water preparing tank, stir, dissolving gets water;
(3) prepare thick emulsion: oil-phase solution is joined aqueous phase, stirring, adding water for injection to overall solution volume is ormal weight, regulates pH value to 4～8.5, continues to stir, and gets thick emulsion;
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, and homogenizing is until reaching suitable granularity requirements;
(5) degerming, fill;
Temperature when wherein said preparation oil phase, water, thick emulsion is 20～80 ℃.
7. docetaxel lyophilization dry lipid microsphere, form by following component:
Docetaxel 1--40g
Oiliness composition 50--500g
Emulsifying agent 5--300g
Stabilizing agent 0--200g
Osmolyte regulator 0--200g
Frozen-dried supporting agent 0--400g
Freeze drying protectant 0-200g
Water for injection adds to 1000ml
8. docetaxel lyophilization dry lipid microsphere according to claim 7, it is characterized in that the oiliness composition is selected from injection soybean oil, safflower oil, medium chain triglycerides, long-chain fatty acid ester, one or more in fractionated oil or modified oil, olive oil, cetylate, Oleum Cocois, Oleum sesami, fish oil, the vitamin E; Emulsifying agent is one or more in soybean phospholipid, lecithin, poloxamer, tween 80, Polyethylene Glycol, cholesterol, oleic acid, benzyl alcohol, ethyl lactate, ethyl oleate, benzyl benzoate, arabic gum, Saponin and the agar.
9. a method for preparing docetaxel lyophilization dry lipid microsphere as claimed in claim 7 comprises the following steps:
(1) preparation oil phase: oiliness composition, emulsifying agent, docetaxel are joined in the oiliness composition preparing tank, attemperation, stir oil phase;
(2) preparation water: with water soluble ingredient, add in the water preparing tank, stir, dissolving gets water;
(3) prepare thick emulsion: oil-phase solution is joined aqueous phase, stirring, adding water for injection to overall solution volume is ormal weight, regulates pH value to 4～8.5, continues to stir, and gets thick emulsion;
(4) preparation lipid microsphere agent: thick emulsion is joined in the high pressure homogenize equipment, and homogenizing is until reaching suitable granularity requirements;
(5) degerming adds proppant, freeze drying protectant, lyophilization, fill;
Temperature when wherein said preparation oil phase, water, thick emulsion is 20～80 ℃.
10. docetaxel lyophilization dry lipid microsphere according to claim 9 is characterized in that proppant and freeze drying protectant are one or more mixture in mannitol, xylitol, sorbitol, glucose, sucrose, glycine, sodium chloride, lactose, citric acid, the hydrolyzed protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101547105A CN1965820A (en) | 2006-11-21 | 2006-11-21 | Lipid microsphere, lyophilized lipid microsphere containing docetaxel and preparation process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101547105A CN1965820A (en) | 2006-11-21 | 2006-11-21 | Lipid microsphere, lyophilized lipid microsphere containing docetaxel and preparation process thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1965820A true CN1965820A (en) | 2007-05-23 |
Family
ID=38074913
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006101547105A Pending CN1965820A (en) | 2006-11-21 | 2006-11-21 | Lipid microsphere, lyophilized lipid microsphere containing docetaxel and preparation process thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1965820A (en) |
-
2006
- 2006-11-21 CN CNA2006101547105A patent/CN1965820A/en active Pending
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Open date: 20070523 |