A kind of taxane liposome lyophilized composition and preparation method thereofTechnical field
The present invention relates to drug preparation technique, relate in particular to a kind of taxane liposome lyophilized composition and preparation method thereof.
Background technology
Paclitaxel was in listing in 1992, and the initial stage is used for the second line treatment of breast carcinoma and uterus carcinoma, share first-line treatment as non-small cell type pulmonary carcinoma and uterus carcinoma in beginning in 1998 and cisplatin.The dosage form of commercially available prod adopts ethanol and Cremophore EL (polyoxyethylene castor oil glycerin ether) drug-supplying system, toxicity greatly, especially anaphylaxis, point out " for preventing severe allergic reaction, all accept this medicine patient should give corticosteroid (as dexamethasone), diphenhydramine and bisfentidine (as cimetidine, ranitidine) in advance " in its description.
------Docetaxel (Docetaxel), its bioavailability and active anticancer all are better than paclitaxel and toxic and side effects is little in to paclitaxel structure of modification process, newly to have synthesized paclitaxel derivant again.Be used for the treatment of breast carcinoma and nonsmall-cell lung cancer through drugs approved by FDA in 1998.Docetaxel also shows ovarian cancer resistance, Kaposi sarcoma, leukemia, colon cancer, melanomatous activity simultaneously.Docetaxel is to be gone on the market with TAXOTERE (docetaxel) injection form by French AVENTIS company the earliest, contain a large amount of Tween80 (polyoxyethylene sorbitan monoleates in the preparation, nonionic surfactant), anaphylaxis is serious, and its medication first three day need give the generation of dexamethasone with the Polyglucan reaction.
Because of taxone is insoluble in water, the taxane drug research of liposome dosage form has become popular research topic.Yet, electroneutral liposome has aggregation tendency, and the film activity effect that hydrophobic taxane had has promoted the liposome gathering, therefore the liposome form of common taxane liposome preparation is very unstable, the bulky grain that reduces, forms because of gathering as envelop rate and taxane is crystalline separates out makes this kind preparation far not reach the security requirement of human vein injecting drug use.
Once some patent disclosures had been arranged and added certain phospholipid component, can improve the report of the stability of liposome, as:
U.S. Pat 5415869 (1995) discloses when adding the electrical phospholipid of bear, be combined into the liposome membrane material as phosphatidyl glycerol ester (PG) and phosphatidylcholine (PC), when paclitaxel accounts for the 3mol% left and right sides of phospholipid, its storage stability is about a week, and medicine carrying then can only be stablized 2 days when reaching 4mol%.
U.S. Pat 5670536 (1997) discloses the Liposomal formulation of a kind of Docetaxel and derivant thereof, this patent points out that with unsaturated phospholipid and a small amount of electrical phospholipid of bear be the combined films material, medicine/when the phospholipid mass ratio is 3-20%, can obtain keeping 8 all clarifying Liposomal formulations in 20 ℃ of storages, its estimation of stability index is that medicine does not have that crystallization is separated out or do not produce precipitation, but does not have particle diameter and envelop rate stability data; In addition, elecrtonegativity phospholipid PG content in lecithin is few, is difficult to obtain, and also has only the reagent specification abroad, costs an arm and a leg.
U.S. Pat 5424073 (1995) and US5648090 (1997) disclose a kind of Paclitaxel liposome prescription, outside main composition PC, add a kind of special phospholipid-------cuorin (cardiolipid), this phospholipid is comparatively special, its molecular structure has a bigger hydrophilic group and 4 fatty carbon chains are formed, can make the paclitaxel of sealing account for the total phospholipids ratio and be promoted to 9mol%, but storing below 4 ℃, its stability is also only about one month, and cuorin is difficult to obtain equally, the reagent specification is also only arranged at present, cost an arm and a leg.
Cost an arm and a leg because these are used to improve stable phospholipid component, they have all limited the marketization of taxanes liposome medicament to a certain extent and have implemented.
Chinese patent CN1291474A has disclosed a kind of Paclitaxel liposome freeze-dried composition and preparation method thereof, composite formula consists of paclitaxel, phospholipid, cholesterol, mannitol or glucose, added aminoacid especially (as lysine, threonine or methionine) as stabilizing agent, described liposome lyophilized composition is the loose block of off-white color, its stability shows as: redissolved after low temperature storage (2-10 degree Celsius) 0-12 month and separate, no crystallization is separated out in its solution, do not assemble, but do not report the particle diameter of liposome composition in solution and the data of envelop rate variation.
Yet, for the antitumor medicinal liposome compositions, if owing to gathering, the fusion of liposome makes its particle diameter in solution greater than more than the 200nm, then its speed that is eliminated in vivo can be accelerated greatly, medicine is eliminated when the no show tumor locus, does not reach the good antitumor effect.
Therefore, improving taxanes liposome drug combination, the especially stability of redissolution dilution back compositions liposome character at low cost, is the technical barrier that those skilled in the art's hope is always for a long time overcome.
Summary of the invention
Technical problem to be solved by this invention be to design a kind of have low cost, redissolve the still stable taxane medicinal liposome freeze-dried composition of back liposome character.
The present invention is in order to solve the problems of the technologies described above, and the solution of the present invention is to add the cyclodextrin composition in the taxane liposome lyophilized composition prescription, and this technology is not seen relevant report, also can't be predicted according to traditional opinion simultaneously.
The invention provides a kind of taxane medicinal liposome freeze-dried composition; it comprises one or more phosphatidylcholines (PC), cholesterol or derivatives thereof, cyclodextrin, taxane and freeze drying protectant; the taxane drug level is 3-6mg/ml in the compositions; compositions has with the dried frozen aquatic products form and is beneficial to its storage stability, and redissolution still can be clarified stabilized liposomes solution after rebuilding.
Taxane liposome lyophilized composition of the present invention, its each component percentage by weight is: taxane 0.4%-1.9%, phospholipid 9.5%-25%, cholesterol 1.5%-2.3%, cyclodextrin 4.5%-1.6%, dried excipient 84.1%-70.2%.
Taxane liposome lyophilized composition of the present invention, its preferred each component percentage by weight is: taxane 0.6%-1.2%, phosphatidase 11 4.5%-22.7%, cholesterol 1.3%-2.1%, cyclodextrin 3.1%-4.7%, freeze-dried excipient 71.6%-78.1%.
Taxane liposome lyophilized composition of the present invention, its more preferred each component percentage by weight is: taxane 0.7%-1.0%, phosphatidase 11 7.0%-20.4%, cholesterol 1.7%-2.1%, cyclodextrin 33%-3.7%, freeze-dried excipient 73.5%-76.4%.
In the taxane liposome lyophilized composition of the present invention, described taxane is paclitaxel or Docetaxel; Described phospholipid can be injection Ovum Gallus domesticus Flavus lecithin, soybean lecithin and/or hydrogenated soy phosphatidyl choline; Described cyclodextrin can be alpha-cyclodextrin, beta-schardinger dextrin-or its derivant separately; Described freeze-dried excipient is sucrose, trehalose, maltose.
Another object of the present invention has provided the preparation method of taxane liposome lyophilized composition, this method is with taxane, phospholipid, cholesterol, cyclodextrin successively stirs in last each component proportioning ratio and is dissolved in dehydrated alcohol or the isopropyl alcohol, make solution become clear and bright solution, put into temperature then and be 30-50 ℃ water bath with thermostatic control, film forming after the removal of solvent under reduced pressure in rotary evaporator, the freeze-dried excipient that adds the 10mM PBS (phosphate buffer) that is dissolved in pH6.8 again, aquation, ultrasonic or homogenizing to particle diameter is about 0.1 μ m, aseptic filtration, divide in the cillin bottle of packing into, lyophilization, promptly get white blocky taxane liposome, vacuum tamponade or logical nitrogen, helium, tamponade behind the argon, sealing.
By the prepared dried frozen aquatic products of technical solution of the present invention, its envelop rate is more than 90%, and mean diameter is in the 60-150 nanometer; Especially redissolving after 5 times of the dilutions envelop rate 8 hours the time with 5% glucose infusion liquid still more than 90%, mean diameter is in the 60-150 nanometer.
Freeze-dried composition of the present invention steady quality still after low temperature (2 ℃-10 ℃) stored 12 months, particularly redissolve dilution back 8h (hour) time still have higher medicinal liposome envelop rate and less liposome particle diameter, thereby both reached the security requirement of human vein injecting drug use, also help improving the therapeutic effect of taxane.Simultaneously, because the present invention does not contain surfactant, so pharmaceutical composition of the present invention is refused patient's antianaphylactic treatment before use, its body can not produce anaphylaxis yet.
The present invention has reached the stability that improves the taxane liposome drug-supplying system, has particularly made liposome lyophilized composition still have the purpose that liposome character is preferably kept in a period of time of redissolving after the dilution.
Find through comparative study, taxane liposome lyophilized composition of the present invention is compared with the taxane liposome lyophilized composition that does not contain the cyclodextrin prescription, under identical preparation method condition, the former not have variation substantially at the liposome encapsulation that redissolves with 5% glucose infusion liquid after 5 times of the dilutions 8 hours the time, the latter's envelop rate then has the reduction of highly significant, and product of the present invention does not still have precipitation when room temperature is deposited 20h separates out, and demonstrates superior quality stability.
Stability after taxane liposome lyophilized composition redissolves relatively
By the taxane liposome lyophilized composition of technical solution of the present invention preparation with 5% glucose infusion liquid redissolve dilute 5 times after, its liposome form is more stable, mean diameter 80 nanometers, the basic no change of envelop rate when room temperature is deposited 8h; And by the taxane liposome that does not contain cyclodextrin (CD) prescription (the same the present invention of all the other components and preparation method, wherein sample No.1 is the Paclitaxel liposome freeze-dried composition) preparation, the envelop rate when room temperature is deposited 8h reduces to 59.6% ± 1.77%; Data see Table 1:
The influence that table 1.CD redissolves the back envelop rate to taxane liposome lyophilized product
| Prescription | Sample | Envelop rate % after lyophilizing is redissolved |
| 0h | 8h |
| Do not contain CD | No.1 No.2 No.3 | 89.9 91.2 93.4 | 58 59.3 61.5 |
| Contain β-CD | Embodiment 3 embodiment 4 embodiment 9 | 91.3 96.1 95.3 | 92.8 96.6 96.3 |
| Contain α-CD | Embodiment 12 embodiment 13 embodiment 14 | 90 92.2 91.3 | 94.7 95.3 96 |
The influence of envelop rate has extremely significant statistical significance (P＜0.001) when containing and not containing cyclodextrin (CD) to taxane liposome lyophilized product redissolution dilution and room temperature placement 8h.
In addition, by the taxane liposome lyophilized composition of technical solution of the present invention preparation, through redissolving dilution 5-160 doubly (Docetaxel concentration can be diluted to 0.01mg/ml), room temperature is deposited more than the 20h, does not still have precipitation and separates out.
Low temperature storage quality stability of the present invention is observed
By the taxane liposome lyophilized composition of technical solution of the present invention preparation, low temperature (2 ℃-10 ℃) store stable aspect also better.Experimental result shows: in 12 months of investigating, the envelop rate of liposome and particle diameter are all basicly stable, and data see Table 2, table 3:
The taxane liposome lyophilized product low temperature storage envelop rate that table 2. contains cyclodextrin changes (%)
| Time (moon) | Embodiment 3 | Embodiment 4 | Embodiment 9 | Embodiment 12 | Embodiment 13 | Embodiment 14 |
| 0 1 3 6 | 91.3 91.8 91.6 91.9 | 93.1 93.5 93.4 93.6 | 95.3 95.1 94.5 94.8 | 93 93.6 93.6 93.3 | 92.2 91.8 92.5 92.1 | 92.5 91.3 92.3 91.8 |
| 12 | 91.1 | 93.5 | 94.5 | 92.5 | 91.5 | 91.2 |
The taxane liposome lyophilized composition that contains cyclodextrin, storage is during 12 months under cryogenic conditions, and envelop rate changes there was no significant difference (P＞0.05).
Table 3. contains the taxane liposome lyophilized product low temperature storage change of size (nm) of cyclodextrin
| Time (moon) | Embodiment 3 | Embodiment 4 | Embodiment 9 | Embodiment 12 | Embodiment 13 | Embodiment 14 |
| 0 1 3 6 12 | 75.7 77.8 77.2 75.3 76.8 | 82.3 80.8 86.3 83.9 89.7 | 79.2 78.6 82.5 81.6 90.3 | 85.3 84.9 86.5 89.1 99.4 | 79.6 82.3 81.5 80.9 95.6 | 84.5 88.2 85.5 86.2 89.9 |
The taxane liposome lyophilized composition that contains cyclodextrin, under cryogenic conditions, store 12 months during, change of size there was no significant difference (P＞0.05).
Thus, taxane liposome lyophilized composition major advantage of the present invention is:
1. have excellent storage quality stability, particularly have superior quality stability in the liquid medium after redissolution, can use for the clinical vein infusion;
2. do not contain surfactant, the patient need not accept the Claritin treatment before medication;
3. the employing of low cost cyclodextrin and hypotoxicity solvent makes the present invention have good market-oriented implementation prospect.
Below in conjunction with specific embodiment, further illustrate the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The method of unreceipted actual conditions in the following example, condition preparation routinely usually.
The specific embodiment
Embodiment 1
The taxane liposome lyophilized composition preparation
Get the 9.5g Ovum Gallus domesticus Flavus lecithin, 1.5g cholesterol, 0.4g Docetaxel, 4.5g HP-β-CD, be dissolved in dehydrated alcohol, place round-bottomed flask, decompression rotary evaporation film forming, the 10mM PBS solution that adds the pH6.8 that contains the 84.1g trehalose, 37 ℃ of aquation 2h are with the ultrasonic or high pressure homogenizer homogenizing of ultrasonoscope, reuse 0.22 μ m membrane filtration degerming, be sub-packed in the cillin bottle, make every bottle to contain the 20mg Docetaxel.Carry out lyophilization, vacuum tamponade or logical noble gas seal and promptly get white block polyene taxol liposome preparation.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 150nm.
Embodiment 2
The taxane liposome lyophilized composition preparation
Get the 25g Ovum Gallus domesticus Flavus lecithin, 1.3g cholesterol, 1.9g Docetaxel, 1.6g HP-β-CD, be dissolved in dehydrated alcohol, place round-bottomed flask, decompression rotary evaporation film forming, the 10mM PBS solution that adds the pH6.8 that contains 70.2g sucrose, 37 ℃ of aquation 2h are with the ultrasonic or high pressure homogenizer homogenizing of ultrasonoscope, reuse 0.22 μ m membrane filtration degerming, be sub-packed in the cillin bottle, make every bottle to contain the 20mg Docetaxel.Carry out lyophilization, vacuum tamponade or logical noble gas seal and promptly get white block polyene taxol liposome preparation.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 60nm.
Embodiment 3
The taxane liposome lyophilized composition preparation
Get the 4g soybean lecithin, 450mg cholesterol, 170mg Docetaxel, 725mg HP-β-CD, be dissolved in dehydrated alcohol, place round-bottomed flask, decompression rotary evaporation film forming, the 10mM PBS solution that adds the pH6.8 that contains 28.8g maltose, 37 ℃ of aquation 2h are with the ultrasonic or high pressure homogenizer homogenizing of ultrasonoscope, reuse 0.22 μ m membrane filtration degerming, be sub-packed in the cillin bottle, make every bottle to contain the 20mg Docetaxel.Carry out lyophilization, vacuum tamponade or logical noble gas seal and promptly get white block polyene taxol liposome preparation.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 80nm.
Embodiment 4
The taxane liposome lyophilized composition preparation
Get the 3.2g Ovum Gallus domesticus Flavus lecithin, 0.8g soybean lecithin, 405mg cholesterol, the 170mg paclitaxel, 870mg HP-β-CD is dissolved in dehydrated alcohol, place round-bottomed flask, decompression rotary evaporation film forming, adding contains the 10mM PBS solution of the pH6.8 of 32g sucrose, 37 ℃ of aquation 2h, ultrasonic or the high pressure homogenizer homogenizing with ultrasonoscope, reuse 0.22 μ m membrane filtration degerming is sub-packed in the cillin bottle, makes every bottle to contain the 20mg Docetaxel.Carry out lyophilization, vacuum tamponade or logical noble gas seal and promptly get white block polyene taxol liposome preparation.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 145nm.
Embodiment 5
The taxane liposome lyophilized composition preparation
Get the 3.6g Ovum Gallus domesticus Flavus lecithin, 0.4g hydrogenated soy phosphatidyl choline, 450mg cholesterol, the 170mg Docetaxel, 870mg HP-β-CD is dissolved in dehydrated alcohol, place round-bottomed flask, decompression rotary evaporation film forming, adding contains the 10mM PBS solution of the pH6.8 of 32g sucrose, 37 ℃ of aquation 2h, ultrasonic or the high pressure homogenizer homogenizing with ultrasonoscope, reuse 0.22 μ m membrane filtration degerming is sub-packed in the cillin bottle, makes every bottle to contain the 20mg Docetaxel.Carry out lyophilization, vacuum tamponade or logical noble gas seal and promptly get white block polyene taxol liposome preparation.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 90.
Embodiment 6
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 5g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg paclitaxel, 870mg HP-β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 120nm.
Embodiment 7
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg Docetaxel, 850mg sulfobutyl ether-β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 90nm.
Embodiment 8
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg Docetaxel, 600mg β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 95nm.
Embodiment 9
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 400mg cholesterol, 170mg paclitaxel, 850mg ethoxy-β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 125nm.
Embodiment 10
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg Docetaxel, 850mg glucosyl group-β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 90nm.
Embodiment 11
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 500mg cholesterol, 170mg Docetaxel, 850mg methyl-β-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 100nm.
Embodiment 12
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 5g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg paclitaxel, 705mg α-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 130nm.
Embodiment 13
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg Docetaxel, 826mg methyl-α-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 120nm.
Embodiment 14
The taxane liposome lyophilized composition preparation
The liposome preparation step is with embodiment 5, and just each component is as follows: 4g Ovum Gallus domesticus Flavus lecithin, 450mg cholesterol, 170mg Docetaxel, 881mg hydroxypropyl-α-CD.Dried frozen aquatic products adds equal volume water for injection jolting dissolving, must clarify the stabilized liposomes suspension, mean diameter 79nm.