CN1236771C - Liposome of 10-hydroxycamptothecine, and its prepn. method - Google Patents
Liposome of 10-hydroxycamptothecine, and its prepn. method Download PDFInfo
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- CN1236771C CN1236771C CN 03113179 CN03113179A CN1236771C CN 1236771 C CN1236771 C CN 1236771C CN 03113179 CN03113179 CN 03113179 CN 03113179 A CN03113179 A CN 03113179A CN 1236771 C CN1236771 C CN 1236771C
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Abstract
The present invention discloses 10-hydroxy camptothecine liposome with a good antitumoral effect, which has favorable water solubility and stability. The therapeutic effects of the 10-hydroxy camptothecine liposome are obviously improved than that of free 10-hydroxy camptothecine. Moreover, the present invention discloses a method for preparing the 10-hydroxy camptothecine liposome. Phospholipid and cholesterol are used as film materials, chloroform or ethanol or ethyl ether is used as a solvent, and mannitol or glucose is used as a freeze-drying protective agent.
Description
Technical field:
The present invention relates to 10-hydroxycamptothecine liposome and preparation method thereof.
Background of invention:
Topoisomerase enzyme inhibitor is the important chemotherapeutics of a class that is used for treatment of cancer, and this compounds has the enzymatic activity effect that suppresses topoisomerase, suppresses topoisomerase and participates in the duplicating of DNA, reparation, genetic recombination and transcribe.10-hydroxycamptothecine is a representative of topoisomerase enzyme inhibitor, and 10-hydroxycamptothecine is a kind of alkaloid that proposes from seed of Nyssaceae deciduous plant Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) or root bark, and Fructus seu radix camptothecae acuminatae (Fructus Camptothecae Acuminatae) is distributed widely in China south, and resource is very abundant.China produces 10-hydroxycamptothecine and all succeeds by chemosynthesis, biofermentation at present.Plant class antineoplastic agent is the leading classification of antitumor clinical application always according to statistics, and 10-hydroxycamptothecine and analog thereof are to become the competitively another kind of plant antineoplastic agent of developmental research of developed country after paclitaxel.China's market share of 10-hydroxycamptothecine in recent years comes front three always in plant class antineoplastic agent.In 10 years, western countries such as American and Britain, method, day successively develop the derivant of a large amount of 10-hydroxycamptothecine classes in the past, and wherein existing part 10-hydroxycamptothecine derivant enters antineoplastic agent market.The mechanism of action of 10-hydroxycamptothecine is very clear and definite, and it is the active chemical compound of a kind of inhibition DNA topoisomerase I.Experimentation shows that it mainly acts on the synthesis stage with DNA, to G
0The phase cell is effect not, to G
1, G
2With M phase cell slight lethality is arranged.It can form a kind of reversible enzyme-10-hydroxycamptothecine-DNA ternary complex, reconnects step during this complex can stop the topoisomerase enzyme reaction to interrupt splitting/to connect to circulate.Zoopery represents that its antitumor spectra is wider, to nucleic acid particularly DNA synthetic the obvious suppression effect is arranged, do not have cross resistance with antitumor drug commonly used.At present clinical being mainly used in treated hepatocarcinoma, colorectal cancer, pulmonary carcinoma and leukemic treatment.But the toxicity of this medicine is bigger, mainly shows as: bone marrow depression, cause leukopenia, gastrointestinal reaction, urinary tract stimulation etc., and these toxicities have limited the clinical practice of 10-hydroxycamptothecine.And because the water-insoluble of 10-hydroxycamptothecine, present clinical formulations employed is to adopt NaOH solution to regulate PH to make Alpha-hydroxy lactonic ring in its structure open and dissolve, but its lactonic ring is a functional group, and the medicine carboxylate form of opening back formation shows as extremely low anti-topoisomerase I activity.
At defective and the limitation of 10-hydroxycamptothecine in clinical practice, can carry out the research transformation of two aspects to 10-hydroxycamptothecine: 1. it is carried out the transformation of chemical constitution, such as the topotecan of abroad having gone on the market, Irinotecan etc., improve water miscible purpose and improve the curative effect effect to reach.2. the employing novel form improves its water solublity and improves curative effect, reduction toxic and side effects, and wherein a kind of method is that it is wrapped in the liposome.Liposome is a kind of bilayer folliculus of similar biofilm structure, and its film material is biodegradation and avirulence and immunogenicity in vivo, thereby can be used as pharmaceutical carrier.Have apolar regions in the liposome, for the intravenously administrable of fat-soluble medicine provides may; Owing to be subjected to the protection of liposome, the medicine that is wrapped can reduce the influence that is subjected to external environment, also can not influence external environment in addition.Because liposome is an exogenous material, enter the picked-up that can be subjected to system of defense in the body in the body, thereby in the normal human, liposome mainly can be in position enrichments such as liver, spleen, lungs, it is dense higher topica to occur, also can play the effect of passive target carrier.
10-hydroxycamptothecine is prepared into liposome can overcomes it because the shortcoming that chemical constitution caused, have following pharmacodynamics and pharmacokinetic characteristics: on the one hand 10-hydroxycamptothecine is prepared into liposome, need not the open loop of Alpha-hydroxy lactonic ring, improved drug effect; It is kept in the neutral aqueous solution, can also avoid the hydrolysis in water environment of Alpha-hydroxy lactonic ring, has improved stability; And because the parcel of liposome can reduce the influence that 10-hydroxycamptothecine produces the hematology; Adopt the liposome dosage form can change interior distribution of body of 10-hydroxycamptothecine on the other hand, make it have liver, lung targeted characteristic.In view of this 2 point, 10-hydroxycamptothecine prepares the toxicity that liposome can reduce medicine, improves the therapeutic effect of medicine simultaneously.
Summary of the invention:
The object of the present invention is to provide a kind of 10-hydroxycamptothecine liposome, do not need to make its dissolving, thereby improved drug effect by the Alpha-hydroxy lactonic ring of opening in the 10-hydroxycamptothecine molecule with good aqueous solubility.
The present invention also aims to provide a kind of preparation to have the method for 10-hydroxycamptothecine liposome good aqueous solubility, stability-enhanced.
Purpose of the present invention can reach by following measure:
A kind of 10-hydroxycamptothecine liposome is characterized in that with following weight proportion be raw material: 10-hydroxycamptothecine 1-2 part, phosphatidase 12 0-150 part, cholesterol 2-15 part.
A kind of 10-hydroxycamptothecine liposome is characterized in that with following weight proportion be raw material: 10-hydroxycamptothecine 1-2 part, phosphatidase 12 0-150 part, cholesterol 2-15 part, mannitol or glucose 25-75 part.
Above-mentioned phospholipid is lecithin, soybean phospholipid or hydrogenated soya phosphatide.
Can comprise aminoacid in the above-mentioned 10-hydroxycamptothecine liposome, aminoacid and 10-hydroxycamptothecine liposome can form complex.
Amino acid whose consumption and 10-hydroxycamptothecine are raw material with following weight proportion: 1 part of 10-hydroxycamptothecine, aminoacid 0.5-3 part.
Above-mentioned aminoacid is selected from glutamic acid, aspartic acid, tyrosine, cysteine.
A kind of preparation method of 10-hydroxycamptothecine liposome, it is characterized in that 10-hydroxycamptothecine, phospholipid, cholesterol are dissolved in chloroform, ethanol or the ether according to the above ratio, constant temperature removes and desolvates, adding is dissolved with the mannitol of aforementioned proportion calculating or the aqueous solution of glucose, and after the dissolving, ultrasound wave or high-pressure homogenization are handled, can obtain the 10-hydroxycamptothecine liposome, be sub-packed in cillin bottle or the ampoule, lyophilization gets the 10-hydroxycamptothecine lipidosome freeze-dried preparation.
A kind of preparation method of 10-hydroxycamptothecine liposome, it is characterized in that 10-hydroxycamptothecine, phospholipid, cholesterol are dissolved in chloroform, ethanol or the ether according to the above ratio, constant temperature removes and desolvates, add the aqueous solution that contains amino acid whose mannitol or glucose that is dissolved with aforementioned proportion calculating, after the dissolving, ultrasound wave or high-pressure homogenization are handled, can obtain the 10-hydroxycamptothecine liposome, be sub-packed in cillin bottle or the ampoule, lyophilization gets the 10-hydroxycamptothecine lipidosome freeze-dried preparation.
It is 40-60 ℃ that above-mentioned constant temperature removes the temperature of desolvating.
Cillin bottle or ampoule feed nitrogen or carbon dioxide after the above-mentioned lyophilization, roll then to cover or seal.
Beneficial effect of the present invention shows:
10-hydroxycamptothecine liposome solutions process lyophilization by the technical solution of the present invention preparation promptly gets the 10-hydroxycamptothecine liposome freeze-drying agent.Can form liposome solutions again through aquation before use, avoid the shortcoming of common liposome, thereby improve the reliability of using with solution form storage stability difference.
10-hydroxycamptothecine liposome freeze-drying agent by the technical solution of the present invention preparation has good water-solubility, no significant change before every index of liposome after the dissolving and the lyophilizing, the concentration of dissolving back 10-hydroxycamptothecine reaches as high as 10mg/ml, requires to dilute according to clinical application; Can avoid simultaneously the hydrolysis of 10-hydroxycamptothecine, improve the stability of 10-hydroxycamptothecine.The 10-hydroxycamptothecine liposome of the present invention's preparation, anticancer therapeutic is compared with commercially available 10-hydroxycamptothecine injection, and drug effect obviously improves.
The inventive method adopts the membrane process preparation, does not adopt the PH gradient method of foreign literature report, and technology is simple.The aminoacid that adds among the present invention can have electric charge under the technical program condition, thereby prevents the liposome gathering, can obviously solve the clustering phenomena of liposome, and is more stable than not adding aminoacid.
The specific embodiment
Embodiment 1
Get 10-hydroxycamptothecine 1.0g, refined lecithin 66g and cholesterol 4.5g put in the round-bottomed flask, add ethanol, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 2g glutamic acid and 50g mannitol, use the Ultrasound Instrument Ultrasonic Pulverization, promptly get the 10-hydroxycamptothecine liposome solutions.The microscopy form detects its particle diameter through the Coulter particle size analyzer.
| TL (mg/ml) | 70.5 |
| Medicine (mg/ml) | 1.0 |
| Mean diameter | 0.7 μ m |
| Encapsulation ratio | 92% |
| Microscopic examination | The liposome particle diameter is very little, and form is good, does not assemble. |
Embodiment 2
Get 10-hydroxycamptothecine 1.0g, refined lecithin 20g and cholesterol 2.0g put in the round-bottomed flask, add chloroform, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 2.0g glutamic acid and 25g mannitol, use the Ultrasound Instrument Ultrasonic Pulverization, promptly get the 10-hydroxycamptothecine liposome solutions.
Embodiment 3
Get 10-hydroxycamptothecine 1.0g, refined lecithin 80g and cholesterol 5.0g put in the round-bottomed flask, add chloroform, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 50g mannitol, use the Ultrasound Instrument Ultrasonic Pulverization, promptly get the 10-hydroxycamptothecine liposome solutions.
Embodiment 4
Get 10-hydroxycamptothecine 1.0g, refined lecithin 100g and cholesterol 5.0g put in the round-bottomed flask, add chloroform, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 1.0g glutamic acid and 50g mannitol, use the Ultrasound Instrument Ultrasonic Pulverization, promptly get the 10-hydroxycamptothecine liposome solutions.
Embodiment 5
Get 10-hydroxycamptothecine 1.0g, refined lecithin 150g and cholesterol 15g put in the round-bottomed flask, add chloroform, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 1.0g glutamic acid and 75g mannitol, use the Ultrasound Instrument Ultrasonic Pulverization, promptly get the 10-hydroxycamptothecine liposome solutions.
Embodiment 6
Get 10-hydroxycamptothecine 1.0g, refining soybean phospholipid 44g and cholesterol 6.0g put in the round-bottomed flask, add chloroform, make said mixture be dissolved into clear and bright solution fully, put water bath with thermostatic control drying under reduced pressure film forming, add the aqueous solution dissolving film that contains 50g mannitol, carry out homogenate with the high-pressure homogenization pump, reuse 0.22 μ m membrane filtration degerming, be sub-packed in cillin bottle or the ampoule, make every bottle to contain 10-hydroxycamptothecine 2mg, lyophilization, logical nitrogen rolls mouth, promptly gets flaxen 10-hydroxycamptothecine lipidosome freeze-dried preparation.
Embodiment 7
Get 10-hydroxycamptothecine liposome and free 10-hydroxycamptothecine solution by embodiment 1 described preparation, the medicine of liposome and free drug all are diluted to required concentration with 5% glucose infusion liquid.
Routinely in mice oxter inoculation hepatocarcinoma HAC, be divided into 7 groups after the inoculation, every group 10, male and female half and half, and in the treatment in 1,3,5 day of inoculation back, treat altogether 3 times, by the body weight administration, difference intravenous injection 10-hydroxycamptothecine liposome 4.0mg/kg, 2.0mg/kg, 1.0mg/kg and free 10-hydroxycamptothecine solution 16.0mg/kg, 8.0mg/kg, 4.0mg/kg and co-content 5% glucose solution; Dissected in back 11 days in inoculation, get the tumor piece calculating tumour inhibiting rate of weighing, and matched group, with relatively doing t check between the dosage, the result shows that 10-hydroxycamptothecine makes the effect that has the inhibition HAC tumor strain of highly significant behind the liposome, the liposome and the commercial preparation of same dosage (4.0mg/kg) have utmost point significant difference, the results are shown in following table:
| Group | Dosage (mg/kg * d) | Number of animals (only) (beginning/end) | Tumour inhibiting rate (%) |
| The liposome 10-hydroxycamptothecine | 4.0×3 | 12/12 | 73.5 |
| 2.0×3 | 12/12 | 60.4 | |
| 1.0×3 | 12/12 | 50.1 | |
| Free 10-hydroxycamptothecine | 16.0×3 | 12/11 | 71.6 |
| 8.0×3 | 12/11 | 56.7 | |
| 4.0×3 | 12/12 | 51.6 |
As above shown in the table, the 10-hydroxycamptothecine therapeutic effect of liposome is better, the dosage that reaches the 10-hydroxycamptothecine of the required liposome of therapeutic effect is 1/4 of free drug dosage, and promptly the therapeutic effect of wrapping kmedicine by liposome is 4 times of free drug at least.
Claims (10)
1, a kind of 10-hydroxycamptothecine liposome is characterized in that with following weight proportion be raw material: 10-hydroxycamptothecine 1-2 part, phosphatidase 12 0-150 part, cholesterol 2-15 part.
2, a kind of 10-hydroxycamptothecine liposome is characterized in that with following weight proportion be raw material: 10-hydroxycamptothecine 1-2 part, phosphatidase 12 0-150 part, cholesterol 2-15 part, mannitol or glucose 25-75 part.
3,10-hydroxycamptothecine liposome according to claim 1 and 2 is characterized in that described phospholipid is lecithin, soybean phospholipid or hydrogenated soya phosphatide.
4,10-hydroxycamptothecine liposome according to claim 1 and 2 wherein can comprise aminoacid, and described aminoacid and 10-hydroxycamptothecine liposome form complex.
5,10-hydroxycamptothecine liposome according to claim 4, wherein amino acid whose consumption and 10-hydroxycamptothecine are raw material with following weight proportion: 1 part of 10-hydroxycamptothecine, aminoacid 0.5-3 part.
6, according to claim 4 or 5 described 10-hydroxycamptothecine liposomees, wherein said aminoacid is selected from glutamic acid, aspartic acid, tyrosine, cysteine.
7, a kind of preparation method of 10-hydroxycamptothecine liposome, it is characterized in that 1-2 part 10-hydroxycamptothecine, 20-150 part phospholipid, 2-15 part cholesterol are dissolved in chloroform, ethanol or the ether, constant temperature removes and desolvates, add the aqueous solution that is dissolved with 25-75 part mannitol or glucose, after the dissolving, ultrasound wave or high-pressure homogenization are handled, can obtain the 10-hydroxycamptothecine liposome, be sub-packed in cillin bottle or the ampoule, lyophilization gets the 10-hydroxycamptothecine lipidosome freeze-dried preparation.
8, the preparation method of 10-hydroxycamptothecine liposome according to claim 7, it is characterized in that 1-2 part 10-hydroxycamptothecine, 20-150 part phospholipid, 2-15 part cholesterol are dissolved in chloroform, ethanol or the ether, constant temperature removes and desolvates, add and be dissolved with 25-75 part mannitol or glucose and the amino acid whose aqueous solution of 0.5-3 part, after the dissolving, ultrasound wave or high-pressure homogenization are handled, can obtain the 10-hydroxycamptothecine liposome, be sub-packed in cillin bottle or the ampoule, lyophilization gets the 10-hydroxycamptothecine lipidosome freeze-dried preparation.
9,, it is characterized in that it is 40-60 ℃ that constant temperature removes the temperature of desolvating according to the preparation method of claim 7 or 8 described 10-hydroxycamptothecine liposomees.
10, according to the preparation method of claim 7 or 8 described 10-hydroxycamptothecine liposomees, it is characterized in that after the lyophilization that cillin bottle or ampoule feed nitrogen or carbon dioxide, roll lid then or seal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03113179 CN1236771C (en) | 2003-04-14 | 2003-04-14 | Liposome of 10-hydroxycamptothecine, and its prepn. method |
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| CN 03113179 CN1236771C (en) | 2003-04-14 | 2003-04-14 | Liposome of 10-hydroxycamptothecine, and its prepn. method |
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| CN1537534A CN1537534A (en) | 2004-10-20 |
| CN1236771C true CN1236771C (en) | 2006-01-18 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326525C (en) * | 2004-11-26 | 2007-07-18 | 复旦大学 | 10-hydroxy camptothecin long cyclic liposome and its freeze aried preparation |
| CN101003556B (en) * | 2007-01-26 | 2012-11-21 | 刘祥华 | Derivative of 10 - hydroxyl camptothecin, prepartion of liposome thereof, and preparation method |
| CN102961335B (en) * | 2012-12-06 | 2014-07-23 | 深圳海王药业有限公司 | Camptothecin medical lipidosome composition and preparation method thereof |
| CN116178474B (en) * | 2023-03-02 | 2024-09-13 | 湖南省中医药研究院 | Camptothecin modified cholesterol and preparation method and application thereof |
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