CN102078317A - Dihydroartemisinine-phospholipid complex and preparation and application thereof - Google Patents
Dihydroartemisinine-phospholipid complex and preparation and application thereof Download PDFInfo
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- CN102078317A CN102078317A CN 201110021972 CN201110021972A CN102078317A CN 102078317 A CN102078317 A CN 102078317A CN 201110021972 CN201110021972 CN 201110021972 CN 201110021972 A CN201110021972 A CN 201110021972A CN 102078317 A CN102078317 A CN 102078317A
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- Prior art keywords
- dihydroarteannuin
- phospholipid
- phosphatide
- phosphatide complexes
- preparation
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- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229930009674 sesquiterpene lactone Natural products 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
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- 238000002560 therapeutic procedure Methods 0.000 description 1
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- 230000006444 vascular growth Effects 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
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Abstract
The invention discloses a dihydroartemisinine-phospholipid complex, a liposome preparation and preparation and application thereof, wherein the dihydroartemisinine-phospholipid complex is a complex prepared from dihydroartemisinine and phospholipid with the mass ratio of 1:(0.1-10); and the liposome preparation is prepared from liposome and a pharmaceutically-acceptable freeze-dried protective agent, wherein the liposome is prepared from a composition comprising the following medicinal raw materials in portion by weight: 0.1 to 10 parts of the dihydroartemisinine-phospholipid complex, and 1 to 80 parts of the phospholipid or 1 to 80 parts of the phospholipid and 0 to 50 parts of cholesterol. The dihydroartemisinine-phospholipid complex can obviously enhance the lypohydrophilic character of the dihydroartemisinine. When the dihydroartemisinine-phospholipid complex is combined with doxorubicin, the cytotoxicity of the doxorubicin can be enhanced and the dosage of the doxorubicin can be reduced, therefore, the toxic side effects of chemotherapeutic medicine in the treatment process can be reduced.
Description
(1) technical field
The present invention relates to a kind of Liposomal formulation and preparation thereof and use particularly a kind of dihydroarteannuin phosphatide complexes and Liposomal formulation and preparation thereof and application.
(2) background technology
Malignant tumor has become the frequently-occurring disease and the commonly encountered diseases of serious threat human life health in the world.According to statistics, there is 1,800,000 people's new trouble malignant tumor every year in China at present, 150 die ten thousand deaths in malignant tumor, and main cause of its treatment failure is the drug resistance that tumor cell produces chemotherapeutics, and malignant tumor tissue the structure function quick hypertrophy of tumor and the transfer that cause unusually.
Dihydroarteannuin (Dihydroartemisinin, DHA) be artemisinin-based drug final active metabolite in vivo, arteannuin be China pharmacy worker 1971 from feverfew Hemerocallis citrina Baroni mugwort extraction separation to a kind of sesquiterpene lactones compounds of peroxide bridge, and use as antimalarial all the time; Along with technological progress and clinical needs, arteannuin with and other pharmacologically actives of derivant obtained paying close attention to widely, especially the antitumor action aspect of arteannuin and derivant thereof, various countries medicine scholar has carried out extensive studies, the mechanism of its antitumor action can be divided into three effects and comprise collaborative, potentiation, the multidrug resistance of reversing tumor, and the effect that suppresses tumor vascular growth and transfer; Collaborative, potentiation: dihydroarteannuin can react with ferrous atom, postpone cell cycle, cell death inducing, as Chinese patent: the dihydroartemisinine of mentioning in the application (application number 200710071185.5) of dihydroartemisinine in strengthening chemotherapy medicine antitumor curative effect is studied with the cyclophosphamide synergistic function, and the combination of artemisinin derivative and vinorelbine does not have and uses the dihydroarteannuin of mentioning in (application number 200810201607.0) and can significantly strengthen vinorelbine to the MCF-7 human breast cancer cell, the A549 human lung carcinoma cell, the cytotoxicity of various kinds of cell such as K562 human leukemia cell; The multidrug resistance of reversing tumor cell: Chinese patent: arteannuin is at the application of preparation in the artitumor multi-medicine-resistant medicine (application number: 200710143393.1) disclose arteannuin and derive and reverse the drug resistance of vincristine drug resistance KB cell; In addition the application (application number 200710071185.5) of dihydroartemisinine in strengthening chemotherapy medicine antitumor curative effect dihydroartemisinine is also disclosed can be by the blocking-up tumor-blood-vessel growth, therefore the fast breeding and the transfer of blocking-up tumor cell can be used as angiogenesis inhibitor and use.These results of study all show, dihydroarteannuin has broad application prospects as the synergist of chemotherapeutics, but dihydroarteannuin is as the derivant of arteannuin, dissolubility in water and soybean oil is all poor, be respectively 0.132 ± 0.016 and 1.63 ± 0.02mg/mL, in the use, especially as synergist and angiogenesis inhibitor use the time, often influence the performance of its drug effect.
The research of phosphatide complexes (phytosomes) is in 1984 reports the earliest by Canadian scientist Venkataram, they find to be dissolved in medicine Griseofulvin and phospholipid in the chloroform jointly, evaporation is removed phospholipid precipitate that organic solvent obtains the stripping of medicine is had obvious facilitation, people have carried out a large amount of research to this dosage form subsequently, breviscapine for example, baicalin, indomethacin etc., these phosphatide complexes can improve the hydrophilic lipophilic character of medicine, improve the dissolution rate of medicine, thereby make the physicochemical property and the biological nature generation change in various degree of chemical compound.
Liposome (liposome) is a kind of new drug delivery system, the small particle diameter liposome can effectively increase medicine circulation time in vivo, strengthen the action effect of medicine, thereby the toxicity of medicine is reduced, toleration improves, consider that dihydroarteannuin is when being used to suppress angiogenic growth, used dosage is bigger, and it is prepared into liposome, to make things convenient for administration, and also there are some researches show to prepare liposome, can improve the envelop rate and the stability of preparation with phosphatide complexes.
(3) summary of the invention
The object of the invention provides a kind of dihydroarteannuin phosphatide complexes and Liposomal formulation, said preparation has improved the hydrophilic lipophilic physicochemical property of dihydroarteannuin, improved dihydroarteannuin toleration in use, the present invention also provides the preparation method and the application in the preparation antitumor drug of dihydroarteannuin phosphatide complexes and Liposomal formulation thereof.
The technical solution used in the present invention is:
A kind of dihydroarteannuin phosphatide complexes, described dihydroarteannuin phosphatide complexes are dihydroarteannuin and the phospholipid complex with mass ratio 1: 0.1～10; Described phospholipid is natural phospholipid or synthetic phospholipid.
Dihydroarteannuin phosphatide complexes of the present invention prepares by the following method: according to proportional quantity, dihydroarteannuin and phospholipid are added in the organic solvent A, 30～60 ℃ of reaction 0.5～4h clarify to reactant liquor, with 30～60 ℃ of vacuum rotary steams of reactant liquor, remove organic solvent, drying promptly gets the dihydroarteannuin phosphatide complexes; The mass ratio of described dihydroarteannuin and phospholipid is 1: 0.1～10, the consumption of described organic solvent A is as the criterion can dissolve dihydroarteannuin and phospholipid and to clarify, consumption does not have much affect, described organic solvent A is one of following: ethyl acetate, oxolane, dichloromethane, chloroform, normal hexane, cyclohexane extraction, methanol, ethanol, isopropyl alcohol, n-butyl alcohol or acetone, described phospholipid are natural phospholipid or synthetic phospholipid.
Described natural phospholipid is soybean lecithin or Ovum Gallus domesticus Flavus lecithin, or the combination of soybean lecithin and Ovum Gallus domesticus Flavus lecithin; Described synthetic phospholipid is distearoyl phosphatidylcholine, distearyl phosphatidyl glycerol, dipalmitoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine or two palmityl PHOSPHATIDYL ETHANOLAMINE, also can be above combination in any.
It is one of following that organic solvent A of the present invention is preferably: ethyl acetate, dichloromethane or acetone.
The present invention also provides a kind of dihydroarteannuin phosphatide complexes prepared dihydroarteannuin phosphatide composite liposome body preparation, the compositions that described preparation is made up of the medicinal raw material of following weight proportion is made liposome, make dihydroarteannuin phosphatide composite liposome body preparation with the acceptable freeze drying protectant of medicine again, described medicinal raw material quality is composed as follows: 0.1～10 part of dihydroarteannuin phosphatide complexes, 1～80 part of phospholipid or 1～80 part of phospholipid and 0～50 part of cholesterol; The mass ratio of dihydroarteannuin and phospholipid is 1: 0.1～10 in the described dihydroarteannuin phosphatide complexes, and phosphorus phosphorus in the described dihydroarteannuin phosphatide complexes and the phospholipid in the described medicinal raw material independently are natural phospholipid or synthetic phospholipid separately.
Described dihydroarteannuin phosphatide composite liposome body preparation, described freeze drying protectant is the combination of following one or more arbitrary proportions: trehalose, lactose, sucrose, mannose or glucose are preferably trehalose, mannose or glucose.
Described dihydroarteannuin phosphatide composite liposome body preparation makes according to following steps:
(1) according to the proportional quantity in the dihydroarteannuin phosphatide complexes, dihydroarteannuin, phospholipid are added in the organic solvent A, under the nitrogen protection, 30～60 ℃ of heating in water bath stir 0.5～2h to the solution clear, the reactant liquor vacuum rotary steam is removed organic solvent A, and drying obtains the dihydroarteannuin phosphatide complexes; Organic solvent A is one of following: ethyl acetate, oxolane, dichloromethane, chloroform, normal hexane, cyclohexane extraction, methanol, ethanol, isopropyl alcohol, n-butyl alcohol or acetone, ethyl acetate, dichloromethane or acetone; (2) phospholipid of prescription amount or the phospholipid and the cholesterol of prescription amount mixed during the dihydroarteannuin phosphatide complexes that step (1) is obtained and medicinal raw material were formed, add organic solvent B, under nitrogen protection, 30～60 ℃ of heating in water bath stir, make the solution clear, gained solution is transferred in the eggplant type bottle, reduction vaporization, remove organic solvent B, and at bottle wall formation layer of transparent thin film, the abundant aquation of buffer solution that adds pH5.0～7.4 is crossed polycarbonate membrane or microjet and is carried out the homogenizing processing, makes dihydroarteannuin phosphatide composite liposome body; Described organic solvent B is one of following: chloroform, methanol or acetone; Described polycarbonate membrane is the preferred 200nm in aperture; (3) the dihydroarteannuin phosphatide composite liposome body that step (2) is made, add freeze drying protectant, and it is fully dissolved, mixing, packing subsequently, lyophilizing promptly gets the dihydroarteannuin lipidosome freeze-dried preparation, and the mass ratio of described dihydroarteannuin phosphatide composite liposome body and freeze drying protectant is 1: 1～20; The adding of described freeze drying protectant does not need heating and stirs, and can be dissolved in the liposome.
In the preparation of described dihydroarteannuin phosphatide composite liposome body preparation, the prepared dihydroarteannuin phosphatide complexes reactant liquor of step (1) also can directly be removed organic solvent, does not carry out the dry phospholipid that directly adds and carries out follow-up reaction.
Step (2) adds the abundant aquation of buffer solution of pH5.0～7.4 in the preparation method of described dihydroarteannuin phosphatide composite liposome body preparation, and described buffer solution is one of following: phosphate buffer, distilled water, normal saline or 5% glucose injection.
Preferred dihydroarteannuin phosphatide composite liposome body preparation, preparation method step (3) is carried out according to following steps: will add freeze drying protectant in the dihydroarteannuin phosphatide composite liposome body, fully dissolving, cross polycarbonate membrane or microjet and carry out the homogenizing processing, make liposome mean diameter≤300nm, packing, lyophilizing promptly gets dihydroarteannuin phosphatide composite liposome body preparation; Described polycarbonate membrane is particle diameter 200nm, continuous film homogenize excessively 10～20 times; The present invention can also microjet carries out the method that homogenizing handles and replaced polycarbonate membrane, and described microjet homogenizing is handled 3 circulations with 5000psi earlier, handles 6～10 circulations with 10000～15000psi then.
Described freeze drying protectant is the combination of following one or more arbitrary proportions: trehalose, mannose or glucose most preferably are mannose.
Organic solvent A of the present invention, organic solvent B are organic solvent, called after organic solvent A, organic solvent B for ease of the organic solvent difference of distinguishing the different step adding, the addition of described organic solvent A, organic solvent B can be dissolved dihydroarteannuin and dihydroarteannuin phosphatide complexes, phospholipid and cholesterol get final product without limits.
The application in the preparation antitumor drug of described dihydroarteannuin phosphatide complexes and Liposomal formulation thereof.
Dihydroarteannuin phosphatide composite liposome body preparation can also be made drug administration by injection or oral administered dosage form with the acceptable accessories carrier.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
The dihydroarteannuin phosphatide complexes can significantly strengthen the hydrophilic lipotropy of dihydroarteannuin, with itself and amycin applied in any combination, can strengthen the cytotoxicity of amycin, reduces the use amount of amycin, thereby reduces the toxic and side effects of chemotherapeutics in therapeutic process; The lipotropy of dihydroarteannuin phosphatide complexes strengthens in addition, also showing as its toxic action to endotheliocyte (HUVEC) further strengthens, compare with the dihydroarteannuin crude drug, its IC50 reduces, toxicity increases, be more suitable for using as angiogenesis inhibitor, it is better to make behind the Liposomal formulation in its body toleration; Simultaneously, when dihydroarteannuin phosphatide composite liposome body preparation of the present invention and amycin coupling, phosphatide complexes can better reverse the drug resistance of amycin persister MCF-7 cell, and dihydroarteannuin phosphatide composite liposome body preparation has significant advantage than dihydroarteannuin.
(4) description of drawings
Fig. 1 is the prepared dihydroarteannuin phosphatide composite liposome body (DHA-LP) of embodiment 1) transmission electron microscope picture;
Fig. 2 is embodiment 10 with embodiment 1 prepared dihydroarteannuin phosphatide complexes (DHA-P) and amycin (DOX) therapeutic alliance lotus S180 sarcoma mouse tumor tumour inhibiting rate;
Fig. 3 is embodiment 11 with embodiment 1 prepared DHA-P and DOX therapeutic alliance B16 melanoma mouse model tumor stereogram: wherein A is blank group, B is the single medication group of DHA, C is the single medication group of DHA-P, D is the single medication group of DOX, E is a DOX+DHA drug combination group, and F is a DOX+DHA-P drug combination group.
(5) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the preparation of dihydroarteannuin phosphoric acid composite
Take by weighing dihydroarteannuin 0.50g, soybean phospholipid 2.56g, add ethyl acetate 40mL, 40 ℃ of reaction 1h clarify to reactant liquor, 30 ℃ of decompression rotary evaporations are removed solvents, and 20 ℃ of vacuum dryings 12 hours promptly get Powdered dihydroarteannuin phosphatide complexes 3.06g, pack, put into 4 ℃ of refrigerators and preserve.
Embodiment 2: the preparation of dihydroarteannuin phosphoric acid composite
Take by weighing dihydroarteannuin 0.50g, distearyl phosphatidyl glycerol 0.50g, add oxolane 40mL, the clarification of 40 ℃ of reaction 2h reactant liquors, 60 ℃ of rotary evaporations of reactant liquor are removed solvent, and 30 ℃ of vacuum dryings 24 hours promptly get Powdered dihydroarteannuin phosphatide complexes 1g, pack, put into 4 ℃ of refrigerators and preserve.
Embodiment 3: the preparation of dihydroarteannuin phosphoric acid composite
Take by weighing dihydroarteannuin 0.50g, soybean lecithin 0.50g adds acetone 20mL, the clarification of 40 ℃ of reaction 4h reactant liquors is removed solvent with the reactant liquor rotary evaporation, 20 ℃ of vacuum dryings 12 hours, promptly get yellow powder powder dihydroarteannuin phosphatide complexes 1g, pack, put into 4 ℃ of refrigerators and preserve.
Embodiment 4: the preparation of dihydroarteannuin phosphoric acid composite
Take by weighing dihydroarteannuin 0.50g, distearyl phosphatidyl glycerol 0.50g, add acetone 20mL, the clarification of 40 ℃ of reaction 4h reactant liquors, the reactant liquor rotary evaporation is removed solvent, and 30 ℃ of vacuum dryings 24 hours promptly get yellow powder powder dihydroarteannuin phosphatide complexes 1g, pack, put into 4 ℃ of refrigerators and preserve.
Embodiment 5: the preparation of dihydroarteannuin phosphoric acid composite Liposomal formulation
Get the prepared dihydroarteannuin phosphatide complexes 1g of embodiment 3; soybean lecithin 5g; cholesterol 2g; add in the 20mL chloroform; under the nitrogen protection; 40 ℃ of heating in water bath stir 1h; make the solution clear; gained solution is transferred in the eggplant-shape bottle; the solvent removed by evaporation at reduced pressure chloroform; and at bottle wall formation layer of transparent thin film; the phosphate buffer 20mL water-fillingization that adds pH7.4; the processing that homogenizes for 10 times of the polycarbonate membrane of crossing 200nm adds mannose 20g subsequently, makes it abundant dissolving; the polycarbonate membrane that the liposome solutions of gained the is crossed 200nm processing that homogenizes for 10 times; packing lyophilizing subsequently promptly gets dihydroarteannuin phosphatide composite liposome body lyophilized formulations 27.9g, particle diameter 256nm.
Embodiment 6: the preparation of dihydroarteannuin phosphoric acid composite Liposomal formulation
Get the prepared dihydroarteannuin phosphatide complexes 0.5g of embodiment 4; soybean lecithin 2g; cholesterol 0.5g; add in the 10mL chloroform; under the nitrogen protection; 40 ℃ of heating in water bath stir 1h; make the solution clear; gained solution is transferred in the eggplant-shape bottle, and reduction vaporization is gone out solvent chloroform, and forms the layer of transparent thin film at the bottle wall; the phosphate buffer 1 0mL water-fillingization that adds pH7.4; cross the microjet processor, microjet is with 5000psi circulation 3 times, then with 10 processing that homogenize of 12000psi circulation; add sucrose 2g subsequently; make it abundant dissolving, the liposome solutions that obtains adds the microjet processor, and microjet is with 5000psi circulation 3 times; then with 10 processing that homogenize of 12000psi circulation; with its packing lyophilizing, promptly get dihydroarteannuin phosphatide composite liposome body lyophilized formulations 4.9g, particle diameter 80nm subsequently.
Embodiment 7: amycin, and dihydroarteannuin, the dihydroarteannuin phosphatide complexes is to MCF-7 amycin sensitive strain cell and the experiment of MCF-7 amycin persister cell inhibiting
1, sample configuration
(1) preparation of medicine liquid: 1. amycin medicinal liquid: get amycin (the chemical industry company limited is completely had an area of in Hainan), add the amycin medicine liquid that the RPMI1640 culture fluid is mixed with 0.05,0.5,1,4,8,50,100 μ g/mL; 2. two arteannuin medicine liquid: dihydroarteannuin (Chongqing Holley Wuling Shan Mountain pharmaceutical Co. Ltd) adds the dihydroarteannuin medicine liquid that the RPMI1640 culture fluid is mixed with 1,2,5,10,20,40,80 μ g/mL; 3. get the prepared dihydroarteannuin phosphatide complexes of embodiment 1, add the RPMI1640 culture fluid, be mixed with the medicine liquid of 1,2,5,10,20,40,80 μ g/mL respectively.
The preparation of the RPMI-1640 of (2) 10% hyclones: 10% hyclone (GIBCO, the U.S.) is added in the RPMI1640 culture fluid (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims bio tech ltd), make the experiment culture fluid.
(3) preparation of 5mg/mLMTT liquid: tetrazole indigo plant (MTT, the general Bioisystech Co., Ltd that flies in Shanghai), the time spent now is mixed with 5mg/mL with normal saline, filtration sterilization, 4 ℃ keep in Dark Place.
2, cytotoxicity experiment
Take the logarithm respectively MCF-7 breast cancer cell amycin sensitive strain (Chinese Academy of Sciences's Shanghai RESEARCH ON CELL-BIOLOGY provides) and the MCF-7 breast cancer cell amycin persister (Chinese Academy of Sciences's Shanghai RESEARCH ON CELL-BIOLOGY provides) of trophophase, make certain density cell suspension with the RPMI-1640 that contains 10% hyclone, add in 96 well culture plates, every porocyte number is 0.5 * 10
4Cultivated 24 hours for 37 ℃, behind the cell attachment, remove culture fluid, the amycin (0.05 that adds variable concentrations respectively, 0.5,1,4,8,50,100 μ g/mL) 100uL, the dihydroarteannuin (1 of variable concentrations, 2,5,10,20,40,80 μ g/mL) the dihydroarteannuin phosphatide complexes (1 of 100uL and variable concentrations, 2,5,10,20,40,80 μ g/mL) medicine liquid 100uL, make blank with culture fluid and not dosing cell, establish six parallel holes for every group, place 37 ℃ of constant incubator (HERAEUS, Germany) cultivate 24h in, every hole adds 5mg/mLMTT liquid 20uL similarity condition and continues to cultivate 4h down, after the supernatant discarded, every hole adds dimethyl sulfoxide (DMSO) 200uL, 37 ℃ of constant incubators are placed 10min, microplate reader (Thermo MK3, the U.S.) detect optical density value (OD value) under the 570nm, experiment repeats 4 times, get average, calculate the variable concentrations amycin according to formula (1), dihydroarteannuin and dihydroarteannuin phosphatide complexes calculate IC to the suppression ratio of MCF-7 breast cancer cell amycin sensitive strain and MCF-7 breast cancer cell amycin persister according to formula (1) inhibitory rate of cell growth
50(being that inhibitory rate of cell growth is 50% o'clock a drug level), each organize numerical value all with
Diagram, P＜0.05 is for difference has statistical significance, and P＜0.01 is remarkable statistical significance for difference has, and the results are shown in Table 1～table 6.
Inhibitory rate of cell growth:
1) amycin is to MCF-7 amycin drug resistance and sensitive strain cell inhibiting rate
The amycin of variable concentrations (DOX) is to the inhibition effect of MCF-7 drug resistance and sensitive strain, the results are shown in Table 1, table 2:
Table 1 amycin is to MCF-7 amycin sensitive strain inhibitory rate of cell growth
Annotate: amycin is 0.221 ± 0.045ug/mL to the IC50 that MCF-7 amycin sensitive strain suppresses
Table 2 amycin is to MCF-7 amycin persister inhibitory rate of cell growth
Annotate: amycin is 47.53 ± 2.53ug/mL to the IC50 that MCF-7 amycin persister suppresses
Table 1 and table 2 as can be seen, according to the IC50 value of amycin to MCF-7 amycin sensitive strain and persister, the drug resistance multiple of amycin persister is about 200 times of sensitive strain.Is benchmark with amycin to the IC50 of amycin persister, just shows when promptly doxorubicin concentration is greater than 47.53ug/mL MCF-7 amycin persister obvious suppression effect comparatively.
2) dihydroarteannuin is to MCF-7 amycin sensitivity and persister cell inhibiting rate
Dihydroarteannuin is to MCF-7 amycin drug resistance and sensitive strain cell inhibiting rate effect, the results are shown in Table 3, table 4.
Table 3 dihydroarteannuin is to MCF-7 amycin sensitive strain inhibitory rate of cell growth
Annotate: dihydroarteannuin is 68.97 ± 2.54ug/mL to the IC50 that MCF-7 amycin sensitive strain suppresses
Table 4 dihydroarteannuin is to MCF-7 amycin persister inhibitory rate of cell growth
Annotate: dihydroarteannuin is 36.35 ± 2.79ug/mL to the IC50 that MCF-7 amycin persister suppresses
From table 3 and table 4 as can be seen, dihydroarteannuin has certain inhibition effect to MCF-7 amycin sensitive strain and persister cell, when the concentration of dihydroarteannuin during, just show obvious inhibition effect to MCF-7 amycin persister and sensitive strain cell respectively greater than 36.35ug/mL and 68.97ug/mL.
3) dihydroarteannuin phosphatide complexes MCF-7 amycin sensitive strain and persister cell inhibiting rate
The dihydroarteannuin phosphatide complexes is to MCF-7 amycin sensitive strain and persister cell inhibiting rate effect, the results are shown in Table 5, table 6.
Table 5 dihydroarteannuin phosphatide complexes is to MCF-7 amycin sensitive strain inhibitory rate of cell growth
Annotate: the dihydroarteannuin phosphatide complexes is 18.13 ± 2.15ug/mL to the IC50 that MCF-7 amycin sensitive strain suppresses
Table 6 dihydroarteannuin phosphatide complexes is to MCF-7 amycin persister inhibitory rate of cell growth
Annotate: the dihydroarteannuin phosphatide complexes is 11.45 ± 1.68ug/mL to the IC50 that MCF-7 amycin persister suppresses
From table 5 and table 6 as can be seen, according to the IC50 value, the dihydroarteannuin phosphatide complexes is compared with the dihydroarteannuin crude drug, the dihydroarteannuin phosphatide complexes has strengthened 3.77 times and 3.27 times respectively to the cytotoxicity of MCF-7 amycin sensitive strain and persister, but this cytotoxicity does not still show selectivity to its cell strain, and promptly the dihydroarteannuin phosphatide complexes does not have difference to the cytotoxicity of MCF-7 amycin sensitive strain and persister.
Embodiment 8: dihydroarteannuin, dihydroarteannuin phosphatide complexes and amycin compositions are to MCF-7 amycin sensitive strain cell and the experiment of MCF-7 amycin persister cell inhibiting
Choosing dihydroarteannuin and dihydroarteannuin phosphatide complexes mixes with amycin (DOX) less than 20% drug level MCF-7 amycin sensitive strain and persister cell inhibitory rate, observation is to MCF-7 amycin sensitive strain and persister cell inhibiting rate, select for use the concentration of dihydroarteannuin to be respectively 24.22ug/mL and 11.78ug/mL, the concentration of dihydroarteannuin phosphatide complexes is respectively: 5.74ug/mL and 2.29ug/mL, mix with variable concentrations DOX respectively, be mixed with dihydroarteannuin amycin (DOX-DHA) medicine, dihydroarteannuin phosphatide complexes amycin (DOX-DHA-P) medicine, other operations the results are shown in Table 7 and table 8 with embodiment 7.
Table 7 dihydroarteannuin and dihydroarteannuin phosphatide complexes mix with amycin MCF-7 sensitive strain inhibitory rate of cell growth
Table 8 dihydroarteannuin and dihydroarteannuin phosphatide complexes mix with amycin MCF-7 persister inhibitory rate of cell growth
Doxorubicin concentration is constant as can be seen from table 7 and table 8, with the MCF-7 cell inhibitory rate is lower than 20% dihydroarteannuin and dihydroarteannuin phosphatide complexes mix use after, it obviously improves MCF-7 cell inhibiting rate, this illustrates that not only dihydroarteannuin has the antitumor potentiation to amycin, and can partly reverse the drug resistance of amycin drug resistance MCF-7 cell.
The dihydroarteannuin phosphatide complexes shows stronger antitumor potentiation in using with mixing of amycin simultaneously, with dihydroarteannuin concentration is that 11.78ug/mL and dihydroarteannuin phosphatide complexes concentration are that 2.29ug/mL mixes with amycin when using, according to the IC50 value, dihydroarteannuin and dihydroarteannuin phosphatide complexes are respectively 8 times and 33 times to the synergic reverse multiple of antitumor, and the dihydroarteannuin phosphatide complexes shows better reverse effect.
Embodiment 9: dihydroarteannuin and dihydroarteannuin phosphatide complexes are tested the HUVEC cell inhibiting
Dihydroarteannuin and dihydroarteannuin phosphatide complexes are to the cytotoxicity of Human umbilical vein endothelial cells HUVEC (the biological company limited of the triumphant base in Nanjing), and experimental implementation is identical with embodiment 7, the results are shown in Table 9,10.
Table 9 dihydroarteannuin is to HUVEC cell inhibiting rate
Annotate: dihydroarteannuin is 25.54 ± 2.59ug/mL to the IC50 of HUVEC cell
Table 10 dihydroarteannuin phosphatide complexes is to HUVEC cell inhibiting rate
Annotate: the dihydroarteannuin phosphatide complexes is 7.74 ± 1.32ug/mL to the IC50 of HUVEC cell
Table 9 and table 10 can be seen, dihydroarteannuin and dihydroarteannuin phosphatide complexes can effectively suppress the growth of endotheliocyte, but the dihydroarteannuin phosphatide complexes can further improve the kill capability of dihydroarteannuin to endotheliocyte, is the effective form of dihydroarteannuin as the tumor vessel inhibitor.
Embodiment 10: the dihydroarteannuin phosphatide complexes strengthens the therapeutical effect of amycin to lotus S180 sarcoma mice
Get ICR mice (the Zhejiang University zoopery center) peritoneal fluid of the lotus S180 sarcoma cell (Shanghai cell institute of the Chinese Academy of Sciences) of cultivating the 7d that goes down to posterity, with normal saline dilution, cell concentration 1 * 10
7Individual/mL is inoculated in the right oxter of mice, and every 0.2mL sets up ICR mice S180 sarcoma oxter inoculation model.To inoculate the back mice and be divided into 7 groups at random, every group 10, numbering is respectively normal saline matched group (NaCl), blank medicine group (Blank) in the group, amycin group (DOX), dihydroarteannuin crude drug group (DHA), dihydroarteannuin phosphatide complexes group (DHA-P), dihydroarteannuin crude drug+amycin group (DOX+DHA), dihydroarteannuin phosphatide complexes+amycin group (DOX+DHA-P), used pair of arteannuin phosphatide complexes is that embodiment 1 is prepared.4d begins the tail vein injection relative medicine after the modeling, every 2d administration 1 time, successive administration 4 times, the each administration 2mg/kg of amycin mice body weight, the each administration 20mg/kg of DHA mice body weight, the DHA-P group, the DOX+DHA group, DOX+DHA-PDOX organizes each dosage and quite contains amycin 2mg/kg mice body weight, and DHA dosage is 20mg/kg mice body weight, record mice body weight every day after the administration, vernier caliper measurement tumor size, at 15d mice is put to death, weigh, peel off tumor and claim its quality, it is heavy that average tumor is respectively organized in calculating, obtain tumor control rate, calculate tumor control rate with formula (3), the result as shown in Figure 2:
Tumour inhibiting rate %=(the average tumor of the average tumor weight/matched group of 1-administration group is heavy) * 100% formula (3)
The result: lotus S180 sarcoma model shows that in the experiment, the compositions of dihydroarteannuin phosphatide complexes and amycin has better tumor killing effect in the body.
Embodiment 11: the dihydroarteannuin phosphatide complexes strengthens the therapeutical effect of amycin to B16 melanoma mice
Choose the ICR mice (Zhejiang University's Experimental Animal Center) of 70 health, be divided into 7 groups at random, every mouse tail vein injection 5 * 10
6Individual B16 melanoma cell is cultivated and is given different preparations respectively after 10 days, and dosage is identical with embodiment 10 with interval, after 4 injections of tail vein, continues routinely to raise, and puts to death the observation pulmonary condition in 30 days behind inoculating cell, the results are shown in Figure 3.
Fig. 3 finds out, Fig. 3-A is that the single medication group of DHA, 3-C are that the single medication group of DHA-P, 3-D are that the single medication group of DOX, 3-E are that DOX+DHA drug combination group, 3-F are DOX+DHA-P drug combination group for blank group, 3-B, B16 melanoma mouse model shows that the pulmonary of mice in the blank group is covered with melanoma cell, the point-like black speck appears in DOX+DHA-P group pulmonary, blank group and DHA+DOX group more all show stronger inhibitory action, DOX+DHA, DOX+DHA-P administration group suppresses effect and is better than independent DOX drug treatment group.
Claims (10)
1. dihydroarteannuin phosphatide complexes is characterized in that described dihydroarteannuin phosphatide complexes is dihydroarteannuin and the phospholipid complex with mass ratio 1: 0.1～10; Described phospholipid is natural phospholipid or synthetic phospholipid.
2. dihydroarteannuin phosphatide complexes as claimed in claim 1, it is characterized in that described complex prepares in accordance with the following methods: according to proportional quantity, dihydroarteannuin and phospholipid are added in the organic solvent A, 30～60 ℃ of reaction 0.5～4h clarify to reactant liquor, with 30～60 ℃ of vacuum rotary steams of reactant liquor, remove organic solvent, drying promptly gets the dihydroarteannuin phosphatide complexes; The mass ratio of described dihydroarteannuin and phospholipid is 1: 0.1～10, described organic solvent A is one of following: ethyl acetate, oxolane, dichloromethane, chloroform, normal hexane, cyclohexane extraction, methanol, ethanol, isopropyl alcohol, n-butyl alcohol or acetone, described phospholipid are natural phospholipid or synthetic phospholipid.
3. dihydroarteannuin phosphatide complexes as claimed in claim 1 is characterized in that described natural phospholipid comprises soybean lecithin and Ovum Gallus domesticus Flavus lecithin; Described synthetic phospholipid comprises distearoyl phosphatidylcholine, distearyl phosphatidyl glycerol, dipalmitoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine or two palmityl PHOSPHATIDYL ETHANOLAMINE.
4. dihydroarteannuin phosphatide complexes as claimed in claim 2 is characterized in that described organic solvent A is one of following: ethyl acetate, dichloromethane or acetone.
5. prepared Liposomal formulation of dihydroarteannuin phosphatide complexes as claimed in claim 1, it is characterized in that the compositions that described Liposomal formulation is made up of the medicinal raw material of following weight proportion makes liposome, make dihydroarteannuin phosphatide composite liposome body preparation with the acceptable freeze drying protectant of medicine again, described medicinal raw material quality is composed as follows: 0.1～10 part of dihydroarteannuin phosphatide complexes, 1～80 part of phospholipid or 1～80 part of phospholipid and 0～50 part of cholesterol; The mass ratio of dihydroarteannuin and phospholipid is 1: 0.1～10 in the described dihydroarteannuin phosphatide complexes, and phospholipid in the described dihydroarteannuin phosphatide complexes and the phospholipid in the described medicinal raw material independently are natural phospholipid or synthetic phospholipid separately.
6. described dihydroarteannuin phosphatide composite liposome body preparation as claimed in claim 5 is characterized in that described freeze drying protectant is the combination of following one or more arbitrary proportions: trehalose, lactose, sucrose, mannose or glucose.
7. dihydroarteannuin phosphatide composite liposome body preparation as claimed in claim 5, it is characterized in that described Liposomal formulation makes according to following steps: (1) is according to proportional quantity, dihydroarteannuin, phospholipid are added in the organic solvent A, under the nitrogen protection, 30～60 ℃ of heating in water bath stir 0.5～2h to the solution clear, the reactant liquor vacuum rotary steam is removed organic solvent A, and drying obtains the dihydroarteannuin phosphatide complexes; Described organic solvent A is one of following: ethyl acetate, oxolane, dichloromethane, chloroform, normal hexane, cyclohexane extraction, methanol, ethanol, isopropyl alcohol, n-butyl alcohol or acetone; (2) the dihydroarteannuin phosphatide complexes that step (1) is obtained mixes with the phospholipid of prescription amount or the phospholipid and the cholesterol of prescription amount, add organic solvent B, under nitrogen protection, 30～60 ℃ of heating in water bath stir, make the solution clear, gained solution is transferred in the eggplant type bottle, reduction vaporization, remove organic solvent B, and at bottle wall formation layer of transparent thin film, the abundant aquation of buffer solution that adds pH5.0～7.4 is crossed polycarbonate membrane or microjet and is carried out the homogenizing processing, makes dihydroarteannuin phosphatide composite liposome body; Described organic solvent B is one of following: chloroform, methanol or acetone; (3) the dihydroarteannuin phosphatide composite liposome body that step (2) is made; add freeze drying protectant; and it is fully dissolved; mixing; packing subsequently; lyophilizing promptly gets the dihydroarteannuin lipidosome freeze-dried preparation, and the mass ratio of described dihydroarteannuin phosphatide composite liposome body and freeze drying protectant is 1: 1～20.
8. dihydroarteannuin phosphatide composite liposome body preparation as claimed in claim 7, it is characterized in that step in the described preparation method (2) adds the abundant aquation of buffer solution of pH5.0～7.4, described buffer solution is one of following: phosphate buffer, distilled water, normal saline or 5% glucose injection.
9. dihydroarteannuin phosphatide composite liposome body preparation as claimed in claim 7, it is characterized in that step in the described preparation method (3) carries out according to following steps: will add freeze drying protectant in the dihydroarteannuin phosphatide composite liposome body, fully dissolving, cross polycarbonate membrane or microjet and carry out the homogenizing processing, make liposome mean diameter≤300nm, packing, lyophilizing promptly gets dihydroarteannuin phosphatide composite liposome body preparation; Described polycarbonate membrane is particle diameter 200nm, continuous film homogenize excessively 10～20 times; It is to handle 3 circulations with 5000psi earlier that described microjet homogenizing is handled, and handles 6～10 circulations with 10000～15000psi then.
10. the application of dihydroarteannuin phosphatide complexes as claimed in claim 1 in the preparation antitumor drug.
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